Cationic polymers for antimicrobial applications and delivery of bioactive materials

ABSTRACT

A cationic star polymer is disclosed of the general formula (1):
 
I′ P′] w′   (1),
 
wherein w′ is a positive number greater than or equal to 3, I′ is a dendritic polyester core covalently linked to w′ independent peripheral linear cationic polymer chains P′. Each of the chains P′ comprises a cationic repeat unit comprising i) a backbone functional group selected from the group consisting of aliphatic carbonates, aliphatic esters, aliphatic carbamates, aliphatic ureas, aliphatic thiocarbamates, aliphatic dithiocarbonates, and combinations thereof, and ii) a side chain comprising a quaternary amine group. The quaternary amine group comprises a divalent methylene group directly covalently linked to i) a positive charged nitrogen and ii) an aromatic ring.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of, and claims the benefitof, Non-Provisional U.S. application Ser. No. 13/077005 entitled“ANTIMICROBIAL COMPOSITIONS, METHODS OF PREPARATION THEREOF, AND USESTHEREOF”, filed on Mar. 31, 2011, herein incorporated by reference inits entirety.

PARTIES TO A JOINT RESEARCH AGREEMENT

This invention was made under a joint research agreement betweenInternational Business Machines Corporation and the Agency For Science,Technology and Research.

BACKGROUND

The invention relates to cationic antimicrobial polymers forantimicrobial applications and delivery of bioactive materials, and morespecifically to unimolecular cationic polycarbonates and/orpolyestercarbonates for antimicrobial applications, gene delivery,and/or drug delivery.

Most conventional antibiotics (e.g., ciprofloxacin, doxycycline andceftazidime) do not physically damage the cell wall but rather penetrateinto the target microorganism and act specifically on targets such asdouble-stranded DNA breakage, inhibition of DNA gyrase, blockage ofmitotic factors or the triggering of intrinsic autolysins. As aconsequence, the bacterial morphology is preserved and the bacteria canreadily develop resistance. In contrast, most cationic peptides (e.g.,magainins, cecropins, protegrins and defensins) do not have a specifictarget in microbes, and interact with the microbial membranes throughelectrostatic interactions, thereby inducing terminal damage tomicrobial membranes.

It has been shown that macromolecular cationic antimicrobial peptidescan overcome bacterial resistance. Most antimicrobial peptides possesscationic and amphiphilic features. Although efforts have been made todesign antimicrobial peptides with various structures over the last twodecades, clinical success has been limited. To date, only four cationicpeptides have successfully entered Phase III clinical trials for woundhealing. This is mainly due to cytotoxicity caused by the cationicnature of peptides (e.g., hemolysis), in vivo short half-life (labile toproteases), and high manufacturing cost.

Amphiphilic biodegradable cationic block copolycarbonates comprisinghydrophilic cationic blocks and hydrophobic blocks are also limited intheir use as antimicrobials. The block copolycarbonate moleculesaggregate in water to form cationic micelles. Although the cationicmicelles are active against Gram-positive bacteria (e.g., Bacillussubtilus), they are less active or non-effective against Gram-negativebacteria (e.g., Escherichia coli). The micelles also de-aggregate atinfinite dilution, which lowers their toxicity to bacteria. Thus, thecritical micelle concentration (CMC) observed with linear blockcopolycarbonates is currently too high for effective systemicadministration of these materials.

Gene therapy holds promise for the treatment of various hereditary andacquired diseases that arise from genetic aberrations. Effective genetherapy requires three separate events. First, the genetic materialwhich is intended to be delivered must be effectively condensed into aparticle having an appropriate size to facilitate extended circulationhalf life. Secondly, the condensed particle must provide protection fromthe host organism's natural defense mechanisms, which are designed toeliminate any foreign genetic material. Finally, the nucleic acids mustbe unpackaged at a desired location allowing their delivery andultimately transcription.

A continuing challenge exists in gene therapy to develop a safe andefficacious vector that can package and protect the genetic material inextracellular environments and penetrate the cell to readily release itsgenetic cargoes. While viral vectors have superior transductioncapabilities, their extensive clinical applications have been greatlylimited by significant immunogenic and carcinogenic risks, costlyproduction, and size restrictions on the encapsulated gene. Of thevarious synthetic transporter materials available, poly(ethylenimine)(PEI) represents a standard for in vitro gene transfection efficiencies.However, the clinical potential of PEI has been drastically limited dueto its non-biodegradability and high cytotoxicity.

Other gene delivery materials, including poly(β-amino esters) (PBAEs),modified PEIs and dendrimers based on poly(amidoamine) (PAMAM) andpoly(L-lysine), are not without unresolved synthetic issues such asrelatively large polydispersities, complex molecular architecturesrequiring multiple production steps, and high cost of starting materials(in the case of amino acids). A narrow molecular weight system isbelieved to be crucial in the clinical settings as individual molecularweight fractions of a polydisperse system are expected to exhibitdistinct pharmacological activities in vivo.

Currently, a growing and urgent need exists for enzymaticallybiodegradable, non-cytotoxic antimicrobial materials that i) exhibithigher toxicity toward a combination of Gram-negative and Gram-positivemicrobes, and ii) disperse in aqueous solution as unimolecularnanostructures having an average particle diameter of 10 nm to 300 nm.This size range is generally suitable for cell wall penetration, andpotentially expands the utility and value of the antimicrobial materialsas multi-use delivery vehicles for genes and/or drugs.

SUMMARY

Accordingly, a cationic star polymer is disclosed having the formula(1):I′

P′]_(w′)  (1),wherein

w′ is a positive number greater than or equal to 3,

I′ is a dendritic polyester core covalently linked to w′ independentperipheral linear cationic polymer chains P′,

each of the chains P′ comprises a cationic repeat unit comprising i) abackbone functional group selected from the group consisting ofaliphatic carbonates, aliphatic esters, aliphatic carbamates, aliphaticureas, aliphatic thiocarbamates, aliphatic dithiocarbonates, andcombinations thereof, and ii) a side chain comprising a quaternary aminegroup, and

the quaternary amine group comprises a divalent methylene group directlycovalently linked to i) a positive charged nitrogen and ii) an aromaticring.

A method of forming the foregoing cationic star polymer is disclosed,which comprises:

forming a mixture containing i) an organocatalyst, ii) an optionalaccelerator, iii) a solvent, iv) a dendritic polyester initiatorcomprising 3 or more peripheral nucleophilic initiator groups for ringopening polymerization (ROP), and v) a cyclic carbonyl monomercomprising a pendant electrophilic group capable of reacting with atertiary amine to form a quaternary amine;

agitating the mixture, thereby forming an electrophilic polymer by ROP;

optionally endcapping the electrophilic polymer, thereby forming anendcapped electrophilic polymer; and

treating the electrophilic polymer and/or the endcapped electrophilicpolymer with the tertiary amine, thereby forming the cationic starpolymer.

Also disclosed is a cationic star polymer comprising: 3 or moreperipheral monovalent linear cationic polymer chains P′ independentlycovalently linked to a dendritic polyester core I′, wherein each of thechains P′ comprises a cationic repeat unit of the general formula (11):

wherein I) t is an integer from 0 to 6, II) each T¹ is a monovalentradical independently selected from the group consisting of hydrogen,and functional groups comprising 1 to 30 carbons, and III) at least oneT¹ group comprises a quaternary amine group, the quaternary amine groupcomprising a divalent methylene group that is directly covalently linkedto i) a positive charged nitrogen and ii) an aromatic ring.

Further disclosed is a cationic graft polymer of formula (1):I′

P′]_(w′)  (1),wherein

w′ is a positive number greater than or equal to 3,

I′ is a core comprising a multivalent linear aliphatic polycarbonate,

I′ is covalently linked to w′ independent monovalent cationic polymerchains P′,

each of the chains P′ comprises a cationic repeat unit comprising i) abackbone functional group selected from the group consisting ofaliphatic carbonates, aliphatic esters, aliphatic carbamates, aliphaticureas, aliphatic thiocarbamates, aliphatic dithiocarbonates, andcombinations thereof, and ii) a side chain comprising a quaternary aminegroup, and

the quaternary amine group comprises a divalent methylene group that isdirectly covalently linked to i) a positive charged nitrogen and ii) anaromatic ring.

A method of forming the foregoing cationic graft polymer is disclosed,comprising:

forming a mixture containing i) an organocatalyst, ii) an optionalaccelerator, iii) a solvent, iv) a cyclic carbonyl monomer comprising apendant electrophilic group capable of reacting with a tertiary amine toform a quaternary amine, and a cyclic carbonyl group selected from thegroup consisting of aliphatic cyclic carbonates, aliphatic cyclicesters, aliphatic cyclic carbamates, aliphatic cyclic ureas, aliphaticcyclic thiocarbamates, aliphatic cyclic dithiocarbonates, andcombinations thereof, and v) an initiator comprising a linear aliphaticpolycarbonate comprising 3 or more side chain nucleophilic groupscapable of initiating a ring opening polymerization (ROP);

agitating the mixture, thereby forming an electrophilic polymer by ROPof the cyclic carbonyl monomer;

optionally endcapping the electrophilic polymer, thereby forming anendcapped electrophilic polymer; and

treating the electrophilic polymer or the endcapped electrophilicpolymer with a tertiary amine, thereby forming the cationic graftpolymer.

Also disclosed is a cationic graft polymer comprising a multivalentlinear aliphatic polycarbonate core I′ comprising 3 or more side chains,the side chains comprising independent monovalent cationic polymerchains P′ covalently linked by respective end units to the side chains,wherein each of the chains P′ comprises a cationic repeat unit of thegeneral formula (11):

wherein I) t is an integer from 0 to 6, II) each T¹ is a monovalentradical independently selected from the group consisting of hydrogen,and groups comprising 1 to 30 carbons, and III) at least one T¹ groupcomprises a quaternary amine group, the quaternary amine groupcomprising a divalent methylene group directly covalently linked to i) apositive charged nitrogen and ii) an aromatic ring.

A method of killing a microbe is disclosed, which comprises contactingthe microbe with any of the foregoing cationic polymers.

An injectable composition is disclosed, which comprises an aqueousmixture of any of the foregoing cationic polymers.

A method of treating a cell is disclosed, which comprises contacting thecell with a composition comprising i) any of the above cationic polymersand ii) a gene and/or a drug.

An article is disclosed which comprises any of the above cationic graftpolymers disposed on a surface of a medical device.

A cationic star polymer is disclosed of formula (1):I′

P′]_(w′)  (1),wherein

w′ is a positive number greater than or equal to 3,

I′ is a multivalent hydrophobic core covalently linked to w′ independentperipheral linear cationic polymer chains P′,

each of the chains P′ comprises a cationic repeat unit comprising i) abackbone functional group selected from the group consisting ofaliphatic carbonates, aliphatic esters, aliphatic carbamates, aliphaticureas, aliphatic thiocarbamates, aliphatic dithiocarbonates, andcombinations thereof, and ii) a side chain comprising a quaternary aminegroup, and

the quaternary amine group comprises a divalent methylene group directlycovalently linked to i) a positive charged nitrogen and ii) an aromaticring.

The above-described and other features and advantages of the presentinvention will be appreciated and understood by those skilled in the artfrom the following detailed description, drawings, and appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graph of % hemolysis as a function of concentration in partsper million (ppm) of cationic star polymers CS-3 (Example 24), CS-6(Example 31), and CS-7 (Example 32) formed from the same electrophilicstar polymer ES-2 having a G′″ core structure (32 arms), and quaternizedwith different amines: trimethylamine (TMA) (CS-3),N,N,N′,N′-tetramethylethylenediamine (TMEDA) (CS-6), andN,N-dimethylethanolamine (DMEA) (CS-7).

FIG. 2 is a graph of % hemolysis as a function of concentration in ppmof cationic graft copolymers CG-1 (Example 25), CG-2 (Example 26), CG-3(comparative Example 27), and CG-4 (Examples 33), each quaternized withtrimethylamine.

FIG. 3 is a black and white photograph of a DNA mobility test atdifferent N/P ratios for a DNA complex of cationic star polymer CS-6(Example 31, 32 arms). The photograph shows a UV illuminated agarose gelplate after gel electrophoresis. The complete retardation of DNAmobility in the gel electrophoresis assay was achieved at N/P ratio of4. N/P ratio is the molar ratio of nitrogen content in the CS-6 polymerto phosphorus in the DNA.

FIG. 4 is a black and white photograph of a DNA mobility test for DNAcomplexes of cationic graft polymers CG-5 (Example 34), CG-6 (Example35), and CG-7 (Example 36). The photograph shows UV illuminated agarosegel plates after gel electrophoresis. The complete retardation of DNAmobility in the gel electrophoresis assay was achieved at N/P ratios of3, 3, and 2, with CG-5, CG-6, and CG-7, respectively.

FIG. 5 is bar chart of luciferase expression in relative light units(RLU) as a function of N/P ratio for a DNA complex of cationic starpolymer CS-6 (Example 31) in HepG2 and 4T1 cell lines. At N/P 40, theefficiency of luciferase expression with CS-6 was comparable to theefficiency of the control PEI at its optimal N/P ratio of 10, a standardfor in vitro gene expression.

FIG. 6 is bar chart of luciferase expression in relative light units(RLU) versus N/P ratio for DNA complexes of cationic graft polymers CG-5(Example 34), CG-6 (Example 35), and CG-7 (Example 36) in HepG2 cellline. Optimal N/P ratios for CG-5, CG-6 and CG-7 were 50, 20 and 20respectively. These cationic graft polymers mediated lower geneexpression efficiency as compared to the cationic star polymer CS-6(Example 31).

FIG. 7 is bar chart of cell viability in percent of HepG2 and 4T1 cellsas a function of N/P ratio of a DNA complex with cationic star polymerCS-6 (Example 31). At the optimal N/P ratio (i.e., N/P=40) for geneexpression, CS-6 (Example 31) was not significantly toxic.

FIG. 8 is bar chart of cell viability in percent of HepG2 cells as afunction of N/P ratio of a DNA complexes made with cationic graftpolymers CG-5 (Example 34), CG-6 (Example 35) and CG-7 (Example 36).More than 80-90% cell viability was achieved with CG-5, CG-6 and CG-7 atN/P=50, 20 and 20, respectively.

DETAILED DESCRIPTION

The antimicrobial cationic polymers of this invention have a structureaccording to general formula (1):I′

P′]_(w′)  (1),wherein w′ is a positive number greater than or equal to 3 and canrepresent an average value. I′ is a multivalent hydrophobic corestructure covalently linked to each of w′ independent monovalentcationic polymer chains P′ by independent divalent linking groups L′ ofI′. That is, the linking groups L′ are represented herein assub-structures of I′. Each of the chains P′ comprises a cationic repeatunit comprising i) an backbone functional group selected from the groupconsisting of aliphatic carbonates, aliphatic esters, aliphaticcarbamates, aliphatic ureas, aliphatic thiocarbamates, aliphaticdithiocarbonates, and combinations thereof, and ii) a side chaincomprising a quaternary amine group. In an embodiment, the cationicrepeat unit comprises an aliphatic carbonate group and/or an aliphaticester group. The quaternary amine group is covalently bound to the sidechain of the cationic repeat unit. The quaternary amine group comprisesa divalent methylene group, which is bonded to i) a positive chargednitrogen and ii) an aromatic ring (e.g., a quaternary nitrogen having abenzyl substituent).

The minimum inhibitory concentration (MIC) of the cationic polymers canbe much less than 500 mg/L against one or more microbes, which caninclude Gram-positive and/or Gram-negative microbes. More particularly,the MIC can be less than 400 mg/L, less than 300 mg/L, or less than 200mg/L against one or more Gram-positive and/or Gram-negative microbes.

Herein, a “cationic star polymer” comprises an aliphatic polyesterdendritic core I′. A “cationic graft polymer” comprises a linearaliphatic polycarbonate or a linear aliphatic polyestercarbonate (i.e.,a polymer having a backbone comprising aliphatic ester repeat units andaliphatic carbonate repeat units) core I′.

In the description that follows, the term “cationic polymer” should beunderstood to mean cationic star polymer and/or cationic graft polymerunless otherwise indicated.

The cationic polymers are amphiphilic, capable of dispersing asunimolecular non-crosslinked nanoparticles in aqueous solution. Thenon-crosslinked cationic polymers are less susceptible to dilutioneffects and have greater antimicrobial activity compared to aqueoussolutions of known non-crosslinked polycarbonates andpolyestercarbonates. The cationic polymers can be used as standaloneantimicrobial agents (i.e., not requiring an additional agent such as apolymer and/or a drug). The cationic polymers exhibit high toxicity toGram-negative microbes, Gram-positive microbes, and yeasts. The cationicpolymers are less cytotoxic compared to the standard reference polymerpoly(ethyleneimine) (PEI) used in gene therapy. Aqueous nanoparticles ofthe non-crosslinked cationic polymers can have an average particlediameter of 50 nm to 250 nm. The cationic polymers can be biocompatibleand/or substantially enzymatically biodegradable. In some cases, acomplex of cationic polymer with a DNA can be as efficient in geneexpression as a complex of PEI with the DNA.

The negative charged counterion of the quaternary amine group can be acovalently bound ionic group of chain P′ (e.g., a side chain carboxylateion), or an ionic group (e.g., chloride ion) that is not covalentlybound to chain P′. In an embodiment, the cationic repeat unit comprisesan aliphatic backbone carbonate group. In another embodiment, thecationic repeat unit comprises an aliphatic backbone ester group. Anoptional non-charged repeat unit of chains P′ can comprise a backbonefunctional group selected from the group consisting of aliphaticcarbonates, aliphatic esters, aliphatic carbamates, aliphatic ureas,aliphatic thiocarbamates, aliphatic dithiocarbonates, and combinationsthereof.

Core structure I′ and/or chains P′ can comprise stereospecific and/ornon-stereospecific repeat units. Core structure I′ is preferablysubstantially or completely non-charged and hydrophobic. Peripheralchains P′ are positive charged and can comprise a hydrophobic repeatunit(s) in the form of a random copolymer or block copolymer structurewith the hydrophilic cationic repeat unit.

Preferably, the dendritic chemical structure of I′ of a cationic starpolymer is essentially free of carboxylic esters of phenolic alcohols,exemplified by the following non-limiting structures:

wherein the starred bonds represent attachment points to other portionsof the chemical structure of I′.

“Essentially free of carboxylic esters of phenolic alcohols” means thatcarboxylic esters of phenolic alcohols are not present in sufficientamount to cause an adverse effect on the antimicrobial properties of thecationic star polymers. In an embodiment, the dendritic chemicalstructure of I′ of the cationic star polymer excludes carboxylic estersof phenolic alcohols. The exclusion arises from the unexpected findingthat cationic star polymers prepared using dendritic ROP initiators,such as D-1 and G-2(OH)₁₂ (below) readily degrade in aqueous and/ororganic protic media. Degradation can occur at low and/or high pH.

By comparison, little or no degradation was observed for G-1(OH)₈, apentaerythritol based dendrimer having no carboxylic ester groups ofphenolic alcohols.

The term “biodegradable” is defined by the American Society for Testingand Materials as a degradation caused by biological activity, especiallyby enzymatic action, leading to a significant change in the chemicalstructure of the material. For purposes herein, a material is“biodegradable” if it undergoes 60% biodegradation within 180 days inaccordance with ASTM D6400. Herein, a material is “enzymaticallybiodegradable” if the material can be degraded (e.g., depolymerized) bya reaction catalyzed by an enzyme.

A “biocompatible” material is defined herein as a material capable ofperforming with an appropriate host response in a specific application.

Herein, a material is “not effective” as an antimicrobial agent if thematerial performs about the same as the phosphate buffered saline (PBS)control solution, and/or has a minimum inhibitory concentration (MIC)greater than 500 mg/L.

Herein, a “stereospecific repeat unit” i) has a non-superposable mirrorimage and ii) comprises one or more asymmetric tetravalent carbons. Eachasymmetric tetravalent carbon is assigned an R or S symmetry based onCahn-Ingold-Prelog (CIP) symmetry rules. Each asymmetric tetravalentcarbon can be present in a stereoisomeric purity of 90% to 100%, 94% ormore, or more particularly 98% to 100%. For example, if thestereospecific repeat unit has two asymmetric tetravalent carbons, thestereospecific repeat unit can be present substantially as the R,Rstereoisomer, substantially as the R,S stereoisomer, substantially asthe S,S stereoisomer, or substantially as the S,R stereoisomer.

A “stereospecific cyclic carbonyl monomer” i) has a non-superposablemirror image and ii) comprises one or more asymmetric tetravalentcarbons. The stereospecific cyclic carbonyl monomer can have astereoisomeric purity of 90% or more, and more particularly 98% or more.In an embodiment, at least one of the asymmetric tetravalent carbons ofthe stereospecific cyclic carbonyl monomer is a ring carbon that becomesa polymer backbone carbon in a ring opening polymerization.

“Restricted metals” herein include ionic and nonionic forms ofberyllium, magnesium, calcium, strontium, barium, radium, aluminum,gallium, indium, thallium, germanium, tin, lead, arsenic, antimony,bismuth, tellurium, polonium, and metals of Groups 3 to 12 of thePeriodic Table. Metals of Groups 3 to 12 of the Periodic Table includescandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium,ruthenium, rhodium, palladium, silver, cadmium, lanthanum, cerium,praseodymium, neodymium, promethium, samarium, europium, gadolinium,terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium,hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold,mercury, actinium, thorium, protactinium, uranium, neptunium, plutonium,americium, curium, berkelium, californium, einsteinium, fermium,mendelevium, nobelium, lawrencium, rutherfordium, dubnium, seaborgium,bohrium, hassium, meitnerium, darmstadtium, roentgenium, andcopernicium. Each one of the foregoing restricted metals can have aconcentration in the cationic polymer of 0 parts to 100 ppm (parts permillion), 0 parts to 100 ppb (parts per billion), or 0 parts to 100 ppt(parts per trillion). Preferably, each one of the foregoing restrictedmetals has a concentration of 0 parts in the cationic polymer (i.e., theconcentration is below detection limits).

No restriction is placed on the concentration of boron, silicon, or anyindividual alkali metal in the cationic polymer, providing theantimicrobial properties of the cationic polymer are not adverselyaffected.

The cationic polymer can be used without an additional biologicallyactive substance (e.g., by applying the cationic polymer as anantimicrobial agent in the form of a liquid solution or a powder to awound surface). Alternatively, the cationic polymer can serve as adelivery vehicle for a biologically active substance that forms acomplex with the cationic polymer by non-covalent interactions. Thiscomplex is referred to as a loaded complex. The loaded complex ispreferably reversible. For example, the loaded complex can enter a cellby endocytosis and release the biologically active substance at adesired stage within the cell or tissues (in the case where the activesubstance is the cell). Biologically active substances include cells,biomolecules (e.g., DNA, genes, peptides, proteins, enzymes, lipids,phospholipids, and nucleotides), natural or synthetic organic compounds(e.g., drugs, dyes, synthetic polymers, oligomers, and amino acids),inorganic materials (e.g., metals and metal oxides), radioactivevariants of the foregoing, and combinations of the foregoing.

“Biologically active” means the referenced material can alter thechemical structure and/or activity of a cell in a desirable manner, orcan selectively alter the chemical structure and/or activity of a celltype relative to another cell type in a desirable manner. As an example,one desirable change in a chemical structure can be the incorporation ofa gene into the DNA of the cell. A desirable change in activity can bethe expression of the transfected gene. Another change in cell activitycan be the induced production of a desired hormone or enzyme.Alternatively, a desirable change in activity can be the selective deathof one cell type over another cell type. No limitation is placed on therelative change in cellular activity caused by the biologically activesubstance, providing the change is desirable and useful. Moreover, nolimitation is placed on the biologically active substance complexed withthe cationic polymer, providing the biologically active substanceinduces a useful cellular response when released from the loadedcomplex.

The following structures illustrate quaternary amines in which adivalent methylene group is directly covalently linked to a positivecharged nitrogen and an aromatic ring (indicated by the arrows):

The starred bonds in the above structures represent attachment points toother portions of the cationic repeat unit. X⁻ is a negative chargedcounterion. In the simplest example, the quaternary nitrogen (positivecharged nitrogen of the quaternary amine) is directly linked to at leastone benzyl group, as shown above. The aromatic ring directly linked tothe divalent methylene group can comprise 0 or more substituents inaddition to the methylene group.

Without being bound by theory, the presence of the aromatic ring inclose proximity to the quaternary nitrogen is believed to enhance theability of the quaternary amine group to bind with and/or penetrate themicrobial cell wall. As the Examples further below demonstrate, thepresence of the aromatic ring increases the antimicrobial activity(i.e., lowers the minimum inhibitory concentration (MIC)) againstGram-negative and Gram-positive microbes compared to otherwisestructurally similar cationic polymers lacking a benzyl substituentlinked to the quaternary nitrogen, such as cationic polymers bearing thefollowing quaternary amine group:

A less preferred convergent synthetic approach for the preparation ofthe cationic polymers comprises i) coupling a preformed electrophilicprecursor chain P′ to a preformed precursor core structure I′ usingsuitable linking chemistry, thereby forming an electrophilic polymer,and ii) treating the electrophilic polymer with a tertiary amine,thereby forming a cationic polymer of formula (1). This convergentsynthetic approach allows for greater functional diversity in thechemical structures and charge densities of the individual chains P′ ofthe cationic polymer. The pre-formed precursor core structure I′ and/orthe preformed electrophilic precursor chain P′ can be prepared byorganocatalyzed ring opening polymerization (ROP).

A preferred sequential method of forming the cationic polymers offormula (1) comprises

i) forming a mixture containing an electrophilic cyclic carbonyl monomercomprising a pendant electrophilic group capable of reacting with atertiary amine to form a quaternary amine, a linear polymeric and/ordendritic ROP initiator (a precursor to core structure I′ in thecationic polymer) comprising 3 or more nucleophilic ROP initiatorgroups, an organocatalyst, an optional accelerator, and a solvent;

ii) agitating the mixture, thereby forming an electrophilic polymer byROP;

iii) optionally endcapping the electrophilic polymer, thereby forming anendcapped electrophilic polymer; and

iv) treating the electrophilic polymer or the endcapped electrophilicpolymer with a tertiary amine, thereby forming a cationic polymer offormula (1).

Scheme 1 illustrates a non-limiting example of a sequential ROP methodfor preparing a cationic star polymer derived from an aliphaticpolyester dendrimer initiator.

Endcapped electrophilic polymer ES-1 is prepared by sequentiallypolymerizing (and ROP-2) of L-lactide, non-charged cyclic ester monomerin ROP-1 followed by MTCOBnCl, an electrophilic cyclic carbonate monomerin ROP-2. ROP-1 is initiated by a first generation aliphatic dendriticpolyol initiator, G-1(OH)₈ (branched aliphatic polyester having 8peripheral hydroxy groups). ROP-2 is initiated by the living end unit ofthe poly(L-lactide) chain formed in ROP-1. ROP-1 and ROP-2 canoptionally be performed in a single pot. Subscripts j and k in the abovestructures of Scheme 1 represent average numbers of repeat units derivedfrom each monomer in each of the precursor chains P′. L-lactide is shownwith R,S-stereochemistry, which is preserved in the ROP. In the abovenon-limiting example, each of the precursor chains P′ of ES-1 comprisesan electrophilic repeat unit comprising a chlorobenzyl side chain group,which is capable of reacting with a tertiary amine (e.g.,trimethylamine) to form a side chain quaternary amine group. In thisinstance, the hydroxyl containing end unit of the electrophilic polymerhas been optionally endcapped as an acetate ester before thequaternization. The endcapped electrophilic polymer ES-1 and/or thenon-endcapped electrophilic polymer can be treated with a tertiary amineto form a cationic star polymer. Reaction of ES-1 with trimethylamineproduces cationic star polymer CS-1 comprising chains P′ having a sidechain quaternary amine group in accordance with formula (1). Thepositive charged nitrogen of the quaternary amine group comprises apara-substituted benzyl moiety. In this example, chains P′ of CS-1 havea block copolymer structure, indicated by the sequential order of thebracketed repeat units in the polymer chain. Each of the divalentoxygens in G′ having a starred bond is a divalent linking group L′ ofI′. The divalent oxygen linking groups L′ are residues of the hydroxyinitiator groups of G1-(OH)₈.

G1-(OH)₈ is an example of a dendritic P′-ROP initiator (i.e., “P′-ROPinitiator” is a term used herein to distinguish the ROP initiator usedto form precursor chains P′). The P′-ROP initiator preferably comprises3 or more, preferably 8 or more nucleophilic ROP initiator groups (e.g.,hydroxy groups). Other examples of dendritic P′-ROP initiators includeG-2(OH)₁₆, and G-3(OH)₃₂:

These and other polyol dendrimers can be prepared by known methods inthe art. The dendritic P′-ROP initiators comprise a plurality ofperipheral nucleophilic end groups capable of initiating a ROP.Preferred dendritic P′-ROP initiators comprise a plurality of branchescomprising repeat units comprising carboxylic esters of aliphaticalcohol groups.

A carboxylic ester of an aliphatic alcohol group comprises an aliphaticcarbon bonded to the ester oxygen, exemplified by the followingnon-limiting examples:

The starred bonds in the above carboxylic esters indicate attachmentpoints to other portions of the compound. The dendritic P′-ROPinitiators are not restricted in the number of generational layers orthe number of branches. The dendritic P′-ROP initiators are preferablyessentially free of carboxylic esters of phenolic alcohols. In anembodiment, the chemical structure of the dendritic P′-ROP initiatorsexcludes carboxylic esters of phenolic alcohols.

The first generation carboxylic ester branches of the dendritic P′-ROPinitiators are covalently linked to a multivalent monomeric core group,referred to herein as a the C′ group, which is preferably derived from amonomeric aliphatic polyol (e.g., pentaerythritol, erythritol, etc.). Inthe above examples of G-1(OH)₈, G-2(OH)₁₆, and G-3(OH)₃₂ the C′ group ofthe dendritic P′-ROP initiator is a pentaerythritolyl group C(CH₂O—*)₄.The first generation dendritic branches of the dendritic P′-ROPinitiator can be prepared by esterification of this monomeric aliphaticpolyol.

Additional examples of C′ groups include the structures of Table 1 andstereoisomers thereof. Asymmetric carbon centers are labeled with R,Sstereochemistry. In Table 1, starred bonds represent potentialattachment points to a dendritic branch of the P′-ROP initiator. One ormore of the oxygens having a starred bond can be individually attachedto an alkyl group such as a methyl and/or ethyl group, as long as atleast three sites of attachment to dendritic branches are present in aC′ group.

TABLE 1

Glycerolyl

Arabitolyl

Threitolyl

Ribitolyl

Xylitolyl

Sorbitolyl

Mannitolyl

Iditolyl

Galactitolyl

Maltitolyl

Isomaltyl

Fructosyl

Lactitolyl

As indicated by the structures in Table 1, the C′ groups of thedendritic P′-ROP initiator can comprise other functional groups, forexample ketones, ketals, acetals, esters, amides, and combinationsthereof.

The nucleophilic initiator groups of the dendritic P′-ROP initiator andother P′-ROP initiators described below can comprise alcohols, primaryamines, secondary amines, thiols, and/or mixtures thereof. PreferredP′-ROP initiators comprise primary alcohol initiator groups.

The electrophilic cyclic carbonyl monomer used in the preparation ofprecursor chains P′ is preferably a cyclic carbonate monomer and/or acyclic ester monomer, having a pendant electrophilic group capable ofreacting with a tertiary amine to form a quaternary amine after the ringopening polymerization (e.g., MTCOPrCl (Table 4 further below) andMTCOBnCl (Scheme 1 above)).

The electrophilic repeat unit of precursor chains P′ preferablycomprises an aliphatic backbone carbonate group and/or an aliphaticbackbone ester group. Precursor chains P′ can comprise a homopolymer,random copolymer, and/or block copolymer chain comprising theelectrophilic repeat unit. Precursor chains P′ can have a living endunit comprising a nucleophilic group capable of initiating a ROP. Asshown in Scheme 1, precursor chains P′ of the electrophilic polymer canoptionally be endcapped.

The P′-ROP initiator can be a linear aliphatic polycarbonate or a linearaliphatic polyestercarbonate polymer comprising a plurality of ROPinitiator groups, referred to herein as a linear polymeric P′-ROPinitiator. The linear polymeric P′-ROP initiator are used to prepare thecationic graft polymers. The linear polymeric P′-ROP initiator comprisesa nucleophilic repeat unit, which has i) a backbone functional groupselected from the group consisting of aliphatic carbonates, aliphaticesters, aliphatic carbamates, aliphatic ureas, aliphatic thiocarbamates,aliphatic dithiocarbonates, and combinations thereof, and ii) a sidechain comprising a nucleophilic ROP initiator group. The nucleophilicrepeat unit preferably comprises an aliphatic backbone carbonate groupor an aliphatic backbone ester group. Each end unit of the linearpolymeric P′-ROP initiator can also comprise a ROP initiator group. Thelinear polymeric P′-ROP initiator comprises 3 or more, preferably 8 ormore, nucleophilic groups capable of initiating a ROP. The linearpolymeric P′-ROP initiator can comprise a homopolymer, random copolymer,and/or block copolymer chain comprising the nucleophilic repeat unit.

PC-1 (see also Example 13 further below) exemplifies a linear polymericP′-ROP initiator (linear polycarbonate) comprising a nucleophilic repeatunit having a backbone aliphatic carbonate group and a side chainaliphatic alcohol:

The vertical stacking of repeat units in PC-1 above indicates a randomdistribution of repeat units in the copolymer chain. In this example,the PC-1 chain is not endcapped. Subscripts m and n in the abovestructure represent the average number of each repeat unit per chain.Using a linear polymeric P′-ROP initiator and organocatalyzed ROP, theprecursor chains P′ can be grown as extensions of the side chainnucleophilic initiator groups and optionally the nucleophilic groups ofthe non-endcapped end units of the linear polymeric P′-ROP initiator,thereby forming an electrophilic graft copolymer (i.e., a precursor to acationic graft copolymer).

EG-1 (below) exemplifies an electrophilic graft copolymer produced inthe above-described manner using the cyclic carbonyl monomers L-lactideand MTCOBnCl:

In the above structure of EG-1, the divalent oxygens having the starredbonds in A′ are divalent linking groups L′ of core structure I′ of EG-1.These divalent oxygens are also divalent linking groups L′ of I′ in thecationic graft polymer formed from EG-1.

In the above example, precursor chains P′ of EG-1 have a block copolymerstructure, indicated by the sequential order of the bracketed repeatunits in the polymer chain. The endcapped or non-endcapped electrophilicpolymer (EG-1) can be treated with a tertiary amine as described furtherabove in a reaction with the chlorobenzyl-containing side chain to forma cationic graft polymer comprising a quaternary amine group (e.g.,Example 25, CG-1). Precursor chain P′ of EG-1 can be performed in asingle pot. Subscripts j and k in the above structure of EG-1 representaverage numbers of repeat units in a precursor chain P′. L-lactide isshown with R,S-stereochemistry, which is preserved in the ROP.

The linear polymeric P′-ROP initiator can also be prepared byorganocatalyzed ROP. In this instance, the ROP utilizes a cycliccarbonyl monomer bearing a pendant protected ROP initiator group,preferably a protected alcohol group, and a mono-nucleophilic ordi-nucleophilic ROP initiator (e.g., mono-alcohol and/or diol). Themono-nucleophilic or di-nucleophilic ROP initiator is referred to hereinas an I′-ROP initiator. The cyclic carbonyl monomer bearing a pendantprotected ROP initiator group comprises i) a cyclic carbonyl functionalgroup selected from the group consisting of cyclic carbonates, cyclicesters, cyclic carbamates, cyclic ureas, cyclic thiocarbamates, cyclicdithiocarbonates, and combinations thereof, and ii) a side chaincomprising a protected nucleophilic ROP initiator group. In anembodiment, the cyclic carbonyl monomer bearing a pendant protected ROPinitiator group, which is used to form the P′-ROP initiator, is selectedfrom the group consisting of a cyclic carbonate monomers, cyclic estermonomers (lactones), and combinations thereof.

A method of forming the linear polymeric P′-ROP initiator comprises i)forming a mixture comprising a cyclic carbonyl monomer bearing aprotected ROP initiator group, an I′-ROP initiator comprising 1 or 2nucleophilic ROP initiator groups, an organocatalyst, an optionalaccelerator, and a solvent, ii) agitating the mixture, thereby forming aprotected linear polymeric P′-ROP initiator, and iii) deprotecting theprotected linear polymeric P′-ROP initiator, thereby forming the linearpolymeric P′-ROP initiator.

The protected linear polymeric P′-ROP initiator comprises a protectedrepeat unit comprising i) a backbone functional group selected from thegroup consisting of aliphatic carbonates, aliphatic esters, aliphaticcarbamates, aliphatic ureas, aliphatic thiocarbamates, aliphaticdithiocarbonates, and combinations thereof, and ii) a side chaincomprising a protected nucleophilic ROP initiator group. The protectedP′-ROP initiator can comprise a homopolymer, random copolymer, and/orblock copolymer chain comprising the protected repeat unit. Scheme 2(see also Example 13) illustrates the preparation of a linear polymericP′-ROP initiator, PC-1.

In this example, the monomeric I′-ROP initiator used in the preparationof PC-1 is BnMPA (benzyl ester of 2,2-bis(methylol)propionic acid),which comprises 2 primary alcohol initiator groups. PC-1 is a linearrandom copolymer produced by ROP of a mixture comprising MTCOBuOBn, acyclic carbonate bearing a pendant benzyl protected primary alcoholgroup, and MTCOEt, a hydrophobic cyclic carbonate comonomer. Palladiumcatalyzed hydrogenolysis of the intermediate protected ROP copolymerdeprotects the benzyl protected side chain alcohol groups, forming thelinear polymeric P′-ROP initiator PC-1. PC-1 comprises a nucleophilicrepeat unit that comprises an aliphatic backbone carbonate group and aside chain alcohol group capable of initiating a ROP.

The above examples illustrate that ring opening polymerizations can beperformed in a stepwise manner to construct the ROP initiators and/orthe electrophilic polymers starting from a monomeric alcohol and/or adiol. Using the sequential approach, core structure I′ of the finalcationic polymer (star or graft) is a residue of the P′-ROP initiator,and w′ represents the number of ROP initiator groups (e.g., hydroxygroups) of the P′-ROP initiator. Each chain P′ is joined to I′ by anindependent divalent linking group L′ comprising a heteroatom residue ofa P′-ROP initiator group (e.g., a divalent oxygen residue of a hydroxyinitiator group, a trivalent nitrogen of an amine, a divalent sulfur ofa thiol). When prepared in this manner, each chain P′ comprises acationic repeat unit derived from the electrophilic cyclic carbonylmonomer.

Although the above-described dendrimer D-1 can effectively initiate aROP, unimolecular aqueous solutions of cationic star polymers preparedfrom D-1 hydrolyze readily in water, and therefore are not preferredantimicrobial agents. Consequently, D-1 is not preferred as a P′-ROPinitiator for preparing the disclosed cationic star polymers having adendritic core structure I′.

Other exclusions can apply to the ROP initiators, cationic starpolymers, and/or cationic graft polymers. The I′-ROP initiator used toform the linear polymeric P′-ROP initiator can be essentially free ofcarboxylate esters of phenolic alcohols. Alternatively, the chemicalstructure of the I′-ROP initiator used to form the linear polymericP′-ROP initiator can exclude carboxylate esters of phenolic alcohols.The linear polymeric P′-ROP initiator used to form the cationic graftpolymer can be essentially free of carboxylate esters of phenolicalcohols. Alternatively, the chemical structure of the linear polymerP′-ROP initiator used to form the cationic graft polymer can excludecarboxylate esters of phenolic alcohols. The chains P′ of the cationicpolymers can be essentially free of carboxylic esters of phenolicalcohols. Alternatively, chains P′ of the disclosed cationic polymerscan exclude carboxylic esters of phenolic alcohols. The cationicpolymers can be essentially free of carboxylic esters of phenolicalcohols. Alternatively, the cationic polymers can exclude carboxylicesters of phenolic alcohols.

Counterions that are not covalently bound to the main chemical frameworkof the cationic polymer can comprise a carboxylic ester of a phenolicalcohol. By “main chemical framework” is meant that portion of thechemical structure of the cationic polymer that contains the majority oflinked covalent bonds (as opposed to unbound counterions).

The main chemical framework of the cationic polymer has a net positivecharge. The net positive charge can result from cationic groups (i.e.,quaternary amine groups) or a mixture of cationic and anionic groups(e.g., carboxylate) that are covalently bound to the main chemicalframework of the cationic polymer. The main chemical framework of thecationic polymer can also comprise latent anionic functional groups thatremain non-charged until contact with a cell.

Also contemplated are cationic polymers comprising sulfonium groups(i.e., a positive charged sulfur bonded to three carbons), andphosphonium groups (i.e., a phosphorous bonded to four carbons (group).The cationic polymer can comprise a mixture of quaternary amine,sulfonium, and phosphonium functional groups.

Non-limiting exemplary negative charged counterions include chloride,bromide, iodide, acetate, benzoate, benzene sulfonate, and toluenesulfonate. The cationic polymer can comprise a mixture of negativecharged counterions.

Polymer chains P′ can further comprise a non-charged second repeat unit.Thus, each polymer chain P′ can independently comprise a homopolymer,random copolymer, or a block copolymer comprising the cationic repeatunit. The cationic repeat unit and/or the non-charged repeat unit can bestereospecific or non-stereospecific.

Cyclic Carbonyl Monomers.

In the following description of cyclic carbonyl monomers, “first cycliccarbonyl monomer” refers to an electrophilic cyclic carbonyl monomercomprising a monovalent leaving group capable of reacting with atertiary amine to form a quaternary amine. “Second cyclic carbonylmonomer” refers to a cyclic carbonyl monomer that can be used as adiluent for the first cyclic carbonyl monomer in order to adjust, forexample, hydrophobicity and/or hydrophilicity of the cationic polymer.The second cyclic carbonyl monomer forms a repeat unit that ishydrophobic and non-charged in the cationic polymer. The first and/orsecond cyclic carbonyl monomer can be stereospecific ornon-stereospecific.

The first cyclic carbonyl monomer and the second cyclic carbonyl monomercan independently be selected from cyclic esters, cyclic carbonates,cyclic carbamates, cylic ureas, cyclic thiocarbamates, cyclicthiocarbonates, and cyclic dithiocarbonates, which have the generalstructures of Table 2.

TABLE 2 Cyclic Ester

Cyclic Carbonate

Cyclic Carbamate

Cyclic Urea

Cyclic Thiocarbamate

Cycl Thiocarbonate

Cyclic Dithiocarbonate

Q¹ in Table 2 is defined under the following formula (2).

More specifically, the first and second cyclic carbonyl monomers can beselected independently from compounds of the general formula (2):

wherein t is an integer from 0 to 6, and when t is 0 carbons labeled 4and 6 are linked together by a single bond. Each Y is a divalent radicalindependently selected from the group consisting of

wherein the starred bond indicates a point of attachment. The latter twogroups are also expressed herein as *—N(Q¹)-* and *—C(Q¹)₂-*. Each Q¹ isa monovalent radical independently selected from the group consisting ofhydrogen, halides, alkyl groups comprising 1 to 30 carbons, aryl groupscomprising 6 to 30 carbon atoms, and groups having the structure

wherein M′ is a monovalent radical selected from the group consisting of*—R¹, *—OR¹, *—N(H)(R¹), *—N(R¹)₂, and *—SR¹, wherein the starred bondrepresents the point of attachment, and each R¹ is a monovalent radicalindependently selected from the group consisting of alkyl groupscomprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons. Afirst cyclic carbonyl monomer of formula (2) comprises one or more Q¹groups comprising a monovalent leaving group capable of reacting with atertiary amine to form a quaternary amine group. A second cycliccarbonyl monomer of formula (2) comprises no monovalent leaving groupthat is capable of reacting with a tertiary amine to form a quaternaryamine.

Non-limiting examples of monovalent leaving groups include halides inthe form of an alkyl halide (e.g., alkyl chloride, alkyl bromide, oralkyl iodide), sulphonate esters (e.g., tosylate or mesylate esters),and epoxides. Each Q¹ group can independently be branched ornon-branched. Each Q¹ group can also independently comprise one or moreadditional functional groups selected from the group consisting ofketones, aldehydes, alkenes, alkynes, cycloaliphatic rings comprising 3to 10 carbons, heterocylic rings comprising 2 to 10 carbons, ethers,amides, esters, and combinations of the foregoing functional groups. Aheterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two ormore Q¹ groups can together form a ring.

The ring opened polymer chain formed with a cyclic carbonyl monomer offormula (2) has a repeat unit having the general formula (3):

wherein Y, t, and Q¹ are defined as above. In an embodiment, Y isoxygen.

A repeat unit of a ring opened polymer formed with a cyclic carbonylmonomer of formula (2) can have a backbone functional group selectedfrom the group consisting of esters, carbonates, ureas, carbamates,thiocarbamates, dithiocarbonates, and combinations thereof, as shown inTable 3.

TABLE 3 Ester

Carbonate

Urea

Carbamate

Thiocarbamate

Thiocarbonate

Dithiocarbonate

wherein Q¹ of Table 3 is defined under formula (2), and R of Table 3 isan aliphatic group

of formula (3) above. Subscript t is defined above under formula (2).

The first and second cyclic carbonyl monomers can be selectedindependently from compounds of the general formula (4):

wherein Q² is a monovalent radical independently selected from the groupconsisting of hydrogen, halides, alkyl groups comprising 1 to 30carbons, aryl groups comprising 6 to 30 carbon atoms, and groups havingthe structure

wherein M′ is a monovalent radical selected from the group consisting of*—R¹, *—OR¹, *—N(H)(R¹), *—N(R¹)₂, and *—SR¹, wherein each R¹ is amonovalent radical independently selected from the group consisting ofalkyl groups comprising 1 to 30 carbons and aryl groups comprising 6 to30 carbons. R² is a monovalent radical independently selected from thegroup consisting of alkyl groups comprising 1 to 30 carbons and arylgroups comprising 6 to 30 carbons, and Q³ is a monovalent radicalselected from the group consisting of hydrogen, alkyl groups having 1 to30 carbons, and aryl groups having 6 to 30 carbons. In an embodiment,each Q² is hydrogen, Q³ is a methyl or ethyl group, and R² is an alkylgroup comprising 1 to 30 carbons. A first cyclic carbonyl monomer offormula (4) comprises an R² group comprising a monovalent leaving groupcapable of reacting with a tertiary amine to form a quaternary amine. Asecond cyclic carbonyl monomer of formula (4) comprises no monovalentleaving group that is capable of reacting with a tertiary amine to forma quaternary amine.

The ring opened polymer chain formed with a cyclic carbonyl monomer offormula (4) has a backbone carbonate repeat unit having the generalformula (5):

wherein Q², Q³, and R² are defined as above.

The first and second cyclic carbonyl monomers can be selected fromcyclic esters of the general formula (6):

wherein u is an integer from 1 to 8, each Q⁴ is a monovalent radicalindependently selected from the group consisting of hydrogen, halides,alkyl groups comprising 1 to 30 carbons, aryl groups comprising 6 to 30carbon atoms, and groups having the structure

where M′ is a monovalent radical selected from the group consisting of*—R¹, *—OR¹, *—N(H)(R¹), *—N(R¹)₂, and *—SR¹, wherein each R¹ is amonovalent radical independently selected from the group consisting ofalkyl groups comprising 1 to 30 carbons and aryl groups comprising 6 to30 carbons. The lactone ring can optionally comprise a carbon-carbondouble bond; that is, optionally, a

group of formula (5) can independently represent a

group. The lactone ring can also comprise a heteroatom such as oxygen,nitrogen, sulfur, or a combination thereof; that is, optionally a

group of formula (6) can independently represent a *—O—*, *—S—*,*—N(H)—*, or an *—N(R¹)—* group, wherein R¹ has the same definition asabove. A first cyclic carbonyl monomer of formula (6) comprises a Q⁴group comprising a monovalent leaving group capable of reacting with atertiary amine to form a quaternary amine. A second cyclic carbonylmonomer of formula (6) comprises no monovalent leaving group capable ofreacting with a tertiary amine to form a moiety comprising a quaternaryamine.

The ring opened polymer chain formed with a cyclic carbonyl monomer offormula (6) has a backbone ester repeat unit having the general formula(7):

wherein Q⁴ and u are defined as above.

The first and/or second cyclic carbonyl monomers can be selected from adioxane dicarbonyl monomers of the general formula (8):

wherein each Q⁵ is a monovalent radical independently selected from thegroup consisting of hydrogen, halides, carboxy groups, alkyl groupscomprising 1 to 30 carbons, aryl groups comprising 6 to 30 carbon atoms,and groups having the structure

where each v is independently an integer from 1 to 6, M′ is a monovalentradical selected from the group consisting of *—R¹, *—OR¹, *—N(H)(R¹),*—N(R¹)₂, and *—SR¹, wherein each R¹ is a monovalent radicalindependently selected from the group consisting of alkyl groupscomprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons,each Q⁶ is a monovalent group independently selected from the groupconsisting of hydrogen, alkyl groups having 1 to 30 carbons, and arylgroups having 6 to 30 carbons. A first cyclic carbonyl monomer offormula (8) comprises a Q⁵ and/or a Q⁶ group comprising a monovalentleaving group capable of reacting with a tertiary amine to form aquaternary amine. A second cyclic carbonyl monomer of formula (8)comprises no monovalent leaving group capable of reacting with atertiary amine to form a moiety comprising a quaternary amine. In anembodiment, the second cyclic carbonyl monomer comprises a compound offormula (8) wherein each v is 1, each Q⁵ is hydrogen, and each Q⁶ is analkyl group comprising 1 to 6 carbons. In an embodiment, the secondcyclic carbonyl monomer is D-lactide or L-lactide.

The ring opened polymer chain formed with a cyclic carbonyl monomer offormula (8) has a backbone ester repeat unit having the general formula(9):

wherein Q⁵, Q⁶, and v are defined as above.

Examples of cyclic carbonate monomers formulas (2) and (4) having amonovalent leaving group in the form of an alkyl halide include thecyclic carbonate monomers of Table 4.

TABLE 4

MTCOPrCl

MTCOPrBr

MTCOEtI

MTCOBnCl

Additional examples of cyclic carbonate monomers of formula (2) and (4)include the compounds of Table 5. These can be used, for example, asdiluent comonomers for the ring-opening polymerization or asintermediates to form other derivatives of cyclic carbonate monomers.

TABLE 5

m = 1: Trimethylene carbonate (TMC) m = 2: Tetramethylene carbonate(TEMC) m = 3: Pentamethylene carbonate (PMC)

R = hydrogen (MTCOH) R = methyl (MTCOMe) R = t-butyl (MTCO^(t)Bu) R =ethyl (MTCOEt)

MTCCl

MTCOBn

MTCTFE

R = methyl R = iso-propyl

MTCOEE

Examples of cyclic carbonyl monomers of formula (6) include thecompounds of Table 6, and stereospecific versions thereof, wherefeasible, comprising one or more stereospecific asymmetric ring carbons.

TABLE 6

R = H; n = 1: beta-Propiolactone (b-PL) R = H; n = 2:gamma-Butyrolactone (g-BL) R = H; n = 3: delta-Valerolactone (d-VL) R =H; n = 4: epsilon-Caprolactone (e-CL) R = CH₃; n = 1: beta-Butyrolactone(b-BL) R = CH₃; n = 2: gamma-Valerolactone (g-VL)

Pivalolactone (PVL)

1,5-Dioxepan-2-one (DXO)

5-(Benzyloxy)oxepan-2-one (BXO)

7-Oxooxepan-4-yl 2-bromo-2-methylpropanoate (BMP-XO)

5-Phenyloxepan-2-one (PXO)

5-Methyloxepan-2-one (MXO)

1,4,8-Trioxa(4,6)spiro-9-undecane (TOSUO)

5-(Benzyloxymethyl)oxepan-2-one (BOMXO)

7-Oxooxepan-4-yl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate(OX-BHMP)

(Z)-6,7-Dihydrooxepin-2(3H)-one (DHXO)

Examples of cyclic carbonyl monomers of formula (8) include thecompounds of Table 7.

TABLE 7

D-Lactide (DLA), L-Lactide (LLA), or racemic Lactide, 1:1 D:L forms(DLLA)

meso-Lactide (MLA) (two opposite centers of asymmetry, R and S)

Glycolide (GLY)

The above monomers can be purified by recrystallization from a solventsuch as ethyl acetate or by other known methods of purification, withparticular attention being paid to removing as much water as possiblefrom the monomer. The monomer moisture content can be from 1 to 10,000ppm, 1 to 1,000 ppm, 1 to 500 ppm, and most specifically 1 to 100 ppm,by weight of the monomer.

I′-ROP Initiators.

The I′-ROP initiator used to prepare the P′-ROP initiator can compriseone or two nucleophilic initiator groups selected from the groupconsisting of alcohols, primary amines, secondary amines, thiols, andcombinations thereof. The I′-ROP initiator can be a monomer, oligomer,or a polymer. The I′-ROP initiator can include other functional groups,including protected nucleophilic groups that include protected thiols,protected amines, and/or protected alcohols. Exemplary monomericmono-nucleophilic I′-ROP initiators include mono-alcohols, such asmethanol, ethanol, propanol, butanol, pentanol, amyl alcohol, caprylalcohol, nonyl alcohol, decyl alcohol, undecyl alcohol, lauryl alcohol,tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol,heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol and otheraliphatic saturated alcohols, cyclopentanol, cyclohexanol,cycloheptanol, cyclooctanol and other aliphatic cyclic alcohols; benzylalcohol, substituted benzyl alcohols, and the like. Exemplary polymericmono-nucleophilic initiators include mono-endcapped poly(ethyleneglycols), and mono-endcapped poly(propylene glycols). Exemplarymonomeric and oligomeric dinucleophilic initiators includebenzenedimethanol, propylene glycol, ethylene glycol, diethylene glycol,and triethylene glycol.

Based on the exclusions mentioned above, phenol, substituted phenols,hydroquinone, resorcinol, and other phenolic compounds are not preferredI′-ROP initiators.

Other dinucleophilic initiators include monomeric diols such as1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, andthe like. As mentioned above, an even more specific dinucleophilicinitiator is BnMPA, a precursor used in the preparation of cycliccarbonate monomers:

A polymeric dinucleophilic initiator can be a polyether diol, morespecifically a poly(alkylene glycol) of the general formula (9):HO—[CH₂(CHR¹)_(x)CHR¹O]_(n)—H  (9),wherein x is 0 or 1, n is an integer from 2 to 10000, each R¹ is amonovalent radical independently selected from the group consisting ofhydrogen and methyl. Thus, the ether repeat unit can comprise 2 or 3backbone carbons between each backbone oxygen. As non-limiting examples,the poly(alkylene glycol) can be a poly(ethylene glycol) (PEG) havingthe structure HO—[CH₂CH₂O]_(n)—H, a poly(propylene glycol) (PPG) havingthe structure HO—[CH₂C(H)(CH₃)O]_(n)—H, or a mixture thereof.

The dinucleophilic polyether initiator can comprise nucleophilic chainend groups independently selected from the group consisting alcohols,primary amines, secondary amines, and thiols. Non-limiting examplesinclude:

One or both end units of the dinucleophilic polyether initiator can bederivatized with substituents having a nucleophilic initiator group forring opening polymerization, as in poly(alkylene oxide)s of generalformula (10):Z′—[CH₂(CHR¹)_(x)CHR¹O]_(n)—Z″  (10)wherein x is 0 or 1, n is an integer from 2 to 10000, each R¹ is amonovalent radical independently selected from the group consisting ofhydrogen and methyl, Z′ is a monovalent radical selected from the groupconsisting of *—OH, *—NH₂, secondary amines, *—SH, and C₁-C₅₀ groupscomprising a nucleophilic initiator group for ring openingpolymerization, Z″ is a monovalent radical selected from the groupconsisting of hydrogen and C₁-C₅₀ groups comprising a nucleophilicinitiator group for ring opening polymerization. At least one of Z′ andZ″ comprises a C₁-C₅₀ group comprising a nucleophilic initiator groupfor ring opening polymerization, the nucleophilic initiator groupselected from the group consisting of alcohols, primary amines,secondary amines, and thiols. In an embodiment, Z′ and/or Z″ comprises abiologically active moiety. In an embodiment x is 0, and each R¹ ishydrogen.

The number average molecular weight of the dinucleophilic polyetherinitiator can be from 100 to 100,000, more specifically 100 to 10000,and even more specifically, 100 to 5000.

Cationic Repeat Units.

In a more specific embodiment, the cationic polymer comprises a cationicrepeat unit of the general formula (11):

wherein i) t is an integer from 0 to 6, and ii) each T¹ is a monovalentradical independently selected from the group consisting of hydrogen,and groups comprising 1 to 30 carbons, and iii) at least one T¹ groupcomprises a quaternary amine group. The quaternary amine group comprisesa divalent methylene group directly bonded to i) a positive chargednitrogen and ii) an aromatic ring.

In an even more specific example, the cationic polymer comprises acationic repeat of the general formula (12):

wherein each T² and T³ are independent monovalent radicals selected fromthe group consisting of hydrogen, and groups comprising 1 to 30 carbons,and T⁴ comprises a quaternary amine group. The quaternary amine groupcomprises a divalent methylene group directly covalently linked to i) apositive charged nitrogen and ii) an aromatic ring. In an embodiment,each T² is hydrogen, and T³ is methyl or ethyl.Endcap Agents.

An endcap agent can prevent further chain growth and stabilize thereactive end groups, minimizing unwanted side reactions (e.g., chainscission). Endcap agents include, for example, materials for convertingterminal hydroxyl groups to esters, such as carboxylic acid anhydrides,carboxylic acid chlorides, or reactive esters (e.g., p-nitrophenylesters). In an embodiment, the endcap agent is acetic anhydride, whichconverts reactive hydroxy end groups to acetate ester groups. The endcapgroup can also be a biologically active moiety.

Quaternization Reaction.

The electrophilic polymer comprises an electrophilic repeat unit derivedfrom the first cyclic carbonyl monomer having a side chain comprising areactive monovalent leaving group capable of reacting with a tertiaryamine to form a quaternary amine. The electrophilic polymer can betreated with a tertiary amine to form the cationic polymer. Thequaternization reaction can be accompanied by minimal, if any,crosslinking of the cationic polymer. The quaternary nitrogen can becovalently linked to the side chain. Alternatively, the quaternarynitrogen can be directly covalently linked to a backbone carbon.

No limitation is placed on the structure of the tertiary amine,providing the resulting quaternary amine group comprises a divalentmethylene group directly covalently linked to i) a positive chargednitrogen and ii) an aromatic ring.

The tertiary amine reacts with more than 0% of the monovalent leavinggroups of the electrophilic polymer to form a quaternary amine,preferably 10% or more, 20% or more, 30% or more, 40% or more, 50% ormore, 60% or more, 70% or more, or more particularly 80% or more of themonovalent leaving groups of the electrophilic polymer.

The tertiary amine can comprise a single nitrogen such as atrialkylamine, including but not limited to trimethylamine,triethylamine, tripropylamine, dimethylbenzylamine, methyldibenzylamine,and the like. The tertiary amine can further comprise additionalfunctional groups, in particular a carboxylic acid group, for example3-(N,N-dimethylamino)propionic acid. In such instances, the cationicpolymer will comprise cationic repeat units comprising a side chaincomprising a quaternary amine group and a carboxylic acid group.

The tertiary amine can also comprise isotopically enriched versions ofthe tertiary amine, such as trimethylamine-¹⁴C, trimethylamine-¹⁵N,trimethylamine-¹⁵N, trimethyl-¹³C₃-amine, trimethyl-d₉-amine, andtrimethyl-d₉-amine-¹⁵N. The tertiary amine can also comprise aradioactive moiety suitable for targeting a specific cell type, such asa cancer cell. The radioactive moiety can comprise a heavy metalradioactive isotope.

In an embodiment, the tertiary amine is a bis-tertiary amine, and thecationic polymer comprises a side chain comprising a quaternary aminegroup and a tertiary amine group. The side chain tertiary amine groupsprovide buffering capacity to facilitate release of the biologicallyactive substance from a loaded complex of the cationic polymer and agene and/or a drug. Bis-tertiary amines have the general formula (13):

wherein L″ is a divalent linking group comprising 2 to 30 carbons, andeach monovalent R^(b) group is independently selected from alkyl groupscomprising 1 to 30 carbons or aryl groups comprising 6 to 30 carbons.Each R^(b) group can independently be branched or non-branched. EachR^(b) group can independently comprise additional functional groups suchas a ketone group, aldehyde group, hydroxyl group, alkene group, alkynegroup, cycloaliphatic ring comprising 3 to 10 carbons, heterocylic ringcomprising 2 to 10 carbons, ether group, amide group, ester group, andcombinations of the foregoing additional functional groups. Theheterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two ormore R^(b) groups can also together form a ring. Representative L″groups include *—(CH₂)_(z′)—* where z′ is an integer from 2 to 30,*—(CH₂CH₂O)_(z*)CH₂CH₂—* where z″ is an integer from 1 to 10,*—CH₂CH₂SCH₂CH₂—*, *—CH₂CH₂SSCH₂CH₂—*, *—CH₂CH₂SOCH₂CH₂—*, and*—CH₂CH₂SO₂CH₂CH₂—*. L″ can further comprise a monovalent or divalentcycloaliphatic ring comprising 3 to 20 carbons, a monovalent or divalentaromatic ring comprising 6 to 20 carbons, a ketone group, aldehydegroup, hydroxyl group, alkene group, alkyne group, a heterocylic ringcomprising 2 to 10 carbons, ether group, amide group, ester group, andcombinations of the foregoing functional groups. The heterocyclic ringcan comprise oxygen, sulfur and/or nitrogen. The bis-tertiary amine canalso comprise isotopically enriched forms of the bis-tertiary amine,such as deuterium, carbon-13, and/or nitrogen-15 enriched forms thereof.

More specific bis-tertiary amines includeN,N,N′,N′-tetramethyl-1,2-ethanediamine (TMEDA),N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPDA),N,N,N′,N′-tetramethyl-1,4-butanediamine (TMBDA),N,N,N′,N′-tetraethyl-1,2-ethanediamine (TEEDA),N,N,N′,N′-tetraethyl-1,3propanediamine (TEPDA),1,4-bis(dimethylamino)cyclohexane, 1,4-bis(dimethylaminobenzene),N,N,N′,N′-tetraethyl-1,4-butanediamine (TEBDA), 4-dimethylaminopyridine(DMAP), 4,4-dipyridyl-1,4-diazabicyclo[2.2.2]octane (DABCO),4-pyrrolidinopyridine, 1-methylbenzimidazole, and combinations thereof.In an embodiment, the bis-tertiary amine is TMEDA.

The electrophilic polymer can be treated with the tertiary amine in asuitable organic solvent (e.g., acetonitrile, dimethylsulfoxide (DMSO),dimethylformamide (DMF), and combinations thereof) to form the cationicpolymer. The reaction is preferably conducted under anhydrousconditions, at ambient or elevated temperature using excess tertiaryamine relative to the monovalent leaving group. In general, the tertiaryamine is used in an amount of from 2 to 30 moles per mole of monovalentleaving group in the electrophilic polymer, more particularly 3 to 20moles per mole of monovalent leaving group in the electrophilic polymer.The positive charged quaternary amine forms a salt with the displacedleaving group, which becomes a negatively charged counterion.Alternatively, the negatively charged counterion can be ion exchangedwith another more suitable negative charged counterion using knownmethods.

The cationic polymer can be isolated by removing excess solvent andamine by vacuum, or by precipitating the cationic polymer in an organicsolvent such as tetrahydrofuran, followed by filtration and drying invacuo. More than 0% of the repeat units derived from the first cycliccarbonyl monomer comprise a side chain moiety comprising a quaternaryamine group. When the electrophilic polymer is treated with abis-tertiary amine, more than 0% of the repeat units derived from thefirst cyclic carbonyl monomer comprise a side chain moiety comprising aquaternary amine group and a tertiary amine group. When theelectrophilic polymer is treated with a tertiary amine comprising acarboxy group or a latent carboxylic acid group, more than 0% of thefirst repeat units derived from the first cyclic carbonyl monomercomprise the side chain moiety comprising the quaternary amine and acarboxylic acid or a latent carboxylic acid group. The quaternary aminegroup is present in the cationic polymer in an amount greater than 0% ofthe side chain monovalent leaving groups derived from the first cycliccarbonyl monomer. More particularly, the quaternary amine group ispresent in the cationic polymer in an amount of 10% to 100%, 20% to100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%,or 80% to 100% of the side chain monovalent leaving groups derived fromthe first cyclic carbonyl monomer.

When the electrophilic polymer is treated with a bis-tertiary amine, thetertiary amine group can be present in the cationic polymer in an amountgreater than 0% of the repeat units comprising a monovalent leavinggroups of the electrophilic polymer, more particularly 10% to 100%, 20%to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to100%, or 80% to 100% of the repeat units comprising a monovalent leavinggroups of the electrophilic polymer.

Ring Opening Polymerizations (ROP).

The following description of methods, conditions and materials for ringopening polymerizations is applicable to the preparation of the P′-ROPinitiator and cationic polymer.

The ring-opening polymerization can be performed at a temperature thatis about ambient temperature or higher, 15° C. to 200° C., and morespecifically 20° C. to 200° C. When the reaction is conducted in bulk,the polymerization is performed at a temperature of 50° C. or higher,and more particularly 100° C. to 200° C. Reaction times vary withsolvent, temperature, agitation rate, pressure, and equipment, but ingeneral the polymerizations are complete within 1 to 100 hours.

The ROP reaction is preferably performed with a solvent. Optionalsolvents include dichloromethane, chloroform, benzene, toluene, xylene,chlorobenzene, dichlorobenzene, benzotrifluoride, petroleum ether,acetonitrile, pentane, hexane, heptane, 2,2,4-trimethylpentane,cyclohexane, diethyl ether, t-butyl methyl ether, diisopropyl ether,dioxane, tetrahydrofuran, or a combination comprising one of theforegoing solvents. When a solvent is present, a suitable monomerconcentration is about 0.1 to 5 moles per liter, and more particularlyabout 0.2 to 4 moles per liter.

The ROP polymerizations are conducted under an inert dry atmosphere,such as nitrogen or argon, and at a pressure of from 100 to 500 MPa (1to 5 atm), more typically at a pressure of 100 to 200 MPa (1 to 2 atm).At the completion of the reaction, the solvent can be removed usingreduced pressure.

Less preferred catalysts for ROP polymerizations include metal oxidessuch as tetramethoxy zirconium, tetra-iso-propoxy zirconium,tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium, tetra-t-butoxyzirconium, triethoxy aluminum, tri-n-propoxy aluminum, tri-iso-propoxyaluminum, tri-n-butoxy aluminum, tri-iso-butoxy aluminum, tri-sec-butoxyaluminum, mono-sec-butoxy-di-iso-propoxy aluminum, ethyl acetoacetatealuminum diisopropylate, aluminum tris(ethyl acetoacetate), tetraethoxytitanium, tetra-iso-propoxy titanium, tetra-n-propoxy titanium,tetra-n-butoxy titanium, tetra-sec-butoxy titanium, tetra-t-butoxytitanium, tri-iso-propoxy gallium, tri-iso-propoxy antimony,tri-iso-butoxy antimony, trimethoxy boron, triethoxy boron,tri-iso-propoxy boron, tri-n-propoxy boron, tri-iso-butoxy boron,tri-n-butoxy boron, tri-sec-butoxy boron, tri-t-butoxy boron,tri-iso-propoxy gallium, tetramethoxy germanium, tetraethoxy germanium,tetra-iso-propoxy germanium, tetra-n-propoxy germanium, tetra-iso-butoxygermanium, tetra-n-butoxy germanium, tetra-sec-butoxy germanium andtetra-t-butoxy germanium; halogenated compound such as antimonypentachloride, zinc chloride, lithium bromide, tin(IV) chloride, cadmiumchloride and boron trifluoride diethyl ether; alkyl aluminum such astrimethyl aluminum, triethyl aluminum, diethyl aluminum chloride, ethylaluminum dichloride and tri-iso-butyl aluminum; alkyl zinc such asdimethyl zinc, diethyl zinc and diisopropyl zinc; heteropolyacids suchas phosphotungstic acid, phosphomolybdic acid, silicotungstic acid andalkali metal salt thereof; zirconium compounds such as zirconium acidchloride, zirconium octanoate, zirconium stearate, and zirconiumnitrate.

The catalyst is preferably an organocatalyst whose chemical formulacontains none of the above-described restricted metals. Examples oforganocatalysts for ring opening polymerizations include tertiary aminessuch as triallylamine, triethylamine, tri-n-octylamine andbenzyldimethylamine 4-dimethylaminopyridine, phosphines, N-heterocycliccarbenes (NHC), bifunctional aminothioureas, phosphazenes, amidines, andguanidines.

A more specific organocatalyst isN-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexyl-thiourea (TU):

Other ROP organocatalysts comprise at least one1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donatinghydrogen bond catalysts have the formula (14):R²—C(CF₃)₂OH  (14),wherein R² represents a hydrogen or a monovalent radical having from 1to 20 carbons, for example an alkyl group, substituted alkyl group,cycloalkyl group, substituted cycloalkyl group, heterocycloalkyl group,substituted heterocycloalklyl group, aryl group, substituted aryl group,or a combination thereof. Exemplary singly-donating hydrogen bondingcatalysts are listed in Table 8.

TABLE 8

4-HFA-St

4-HFA-Tol

HFTB

NFTB

HFIP

Doubly-donating hydrogen bonding catalysts have two HFP groups,represented by the general formula (15):

wherein R³ is a divalent radical bridging group containing from 1 to 20carbons, such as an alkylene group, a substituted alkylene group, acycloalkylene group, substituted cycloalkylene group, aheterocycloalkylene group, substituted heterocycloalkylene group, anarylene group, a substituted arylene group, and a combination thereof.Representative double hydrogen bonding catalysts of formula (15) includethose listed in Table 9. In a specific embodiment, R² is an arylene orsubstituted arylene group, and the HFP groups occupy positions meta toeach other on the aromatic ring.

TABLE 9

3,5-HFA-MA

3,5-HFA-St

1,3-HFAB

1,4-HFAB

In one embodiment, the catalyst is selected from the group consisting of4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA, 3,5-HFA-St, 1,3-HFAB,1,4-HFAB, and combinations thereof.

Also contemplated are catalysts comprising HFP-containing groups boundto a support. In one embodiment, the support comprises a polymer, acrosslinked polymer bead, an inorganic particle, or a metallic particle.HFP-containing polymers can be formed by known methods including directpolymerization of an HFP-containing monomer (for example, themethacrylate monomer 3,5-HFA-MA or the styryl monomer 3,5-HFA-St).Functional groups in HFP-containing monomers that can undergo directpolymerization (or polymerization with a comonomer) include acrylate,methacrylate, alpha, alpha, alpha-trifluoromethacrylate,alpha-halomethacrylate, acrylamido, methacrylamido, norbornene, vinyl,vinyl ether, and other groups known in the art. Examples of linkinggroups include C₁-C₁₂ alkyl, a C₁-C₁₂ heteroalkyl, ether group,thioether group, amino group, ester group, amide group, or a combinationthereof. Also contemplated are catalysts comprising chargedHFP-containing groups bound by ionic association to oppositely chargedsites on a polymer or a support surface.

The ROP reaction mixture comprises at least one organocatalyst and, whenappropriate, several organocatalysts together. The ROP catalyst is addedin a proportion of 1/20 to 1/40,000 moles relative to the cycliccarbonyl monomers, and preferably in a proportion of 1/1,000 to 1/20,000moles relative to the cyclic carbonyl monomers.

ROP Accelerators.

The ROP polymerization can be conducted in the presence of an optionalaccelerator, in particular a nitrogen base. Exemplary nitrogen baseaccelerators are listed below and include pyridine (Py),N,N-dimethylaminocyclohexane (Me₂NCy), 4-N,N-dimethylaminopyridine(DMAP), trans 1,2-bis(dimethylamino)cyclohexane (TMCHD),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (−)-sparteine, (Sp)1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1),1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2),1,3-bis(2,6-di-1-propylphenyl(imidazol-2-ylidene (Im-3),1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4),1,3-di-1-propylimidazol-2-ylidene (Im-5),1,3-di-t-butylimidazol-2-ylidene (Im-6),1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7),1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene,1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8) or acombination thereof, shown in Table 10.

TABLE 10

Pyridine (Py)

N,N-Dimethylaminocyclohexane (Me₂NCy)

4-N,N-Dimethylaminopyridine (DMAP)

trans 1,2-Bis(dimethylamino)cyclohexane (TMCHD)

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)

7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD)

1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD)

(−)-Sparteine (Sp)

1,3-Bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1)

1,3-Bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2)

1,3-Bis(2,6-di-i-propylphenyl(imidazol-2-ylidene (Im-3)

1,3-Bis(1-adamantyl)imidazol-2-yliden) (Im-4)

1,3-Di-i-propylimidazol-2-ylidene (Im-5)

1,3-Di-t-butylimidazol-2-ylidene (Im-6)

1,3-Bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7)

1,3-Bis(2,6-di-i-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8)

In an embodiment, the accelerator has two or three nitrogens, eachcapable of participating as a Lewis base, as for example in thestructure (−)-sparteine. Stronger bases generally improve thepolymerization rate.

The catalyst and the accelerator can be the same material. For example,some ring opening polymerizations can be conducted using1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) alone, with no another catalystor accelerator present.

The catalyst is preferably present in an amount of about 0.2 to 20 mol%, 0.5 to 10 mol %, 1 to 5 mol %, or 1 to 2.5 mol %, based on totalmoles of cyclic carbonyl monomer.

The nitrogen base accelerator, when used, is preferably present in anamount of 0.1 to 5.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2to 0.5 mol %, based on total moles of cyclic carbonyl monomer. As statedabove, in some instances the catalyst and the nitrogen base acceleratorcan be the same compound, depending on the particular cyclic carbonylmonomer.

The amount of initiator is calculated based on the equivalent molecularweight per nucleophilic initiator group in the ROP initiator. Theinitiator groups are preferably present in an amount of 0.001 to 10.0mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, and 0.2 to 0.5 mol %, basedon total moles of cyclic carbonyl monomer used in the polymerization.For example, if the molecular weight of the initiator is 100 g/mole andthe initiator has 2 hydroxyl groups, the equivalent molecular weight perhydroxyl group is 50 g/mole. If the polymerization calls for 5 mol %hydroxyl groups per mole of monomer, the amount of initiator is0.05×50=2.5 g per mole of monomer.

In a specific embodiment, the catalyst is present in an amount of about0.2 to 20 mol %, the nitrogen base accelerator is present in an amountof 0.1 to 5.0 mol %, and the nucleophilic initiator groups of theinitiator are present in an amount of 0.1 to 5.0 mol % based on theequivalent molecular weight per nucleophilic initiator group of theinitiator.

The catalysts can be removed by selective precipitation or in the caseof the solid supported catalysts, simply by filtration. The product ofthe ROP can comprise residual catalyst in an amount greater than 0 wt. %(weight percent), based on total weight of the block copolymer and theresidual catalyst. The amount of residual catalyst can also be less than20 wt. %, less than 15 wt. %, less than 10 wt. %, less than 5 wt. %,less than 1 wt. %, or most specifically less than 0.5 wt. % based on thetotal weight of the ROP polymer and the residual catalyst.

Average Molecular Weight.

The cationic polymer and/or the electrophilic polymer preferably has anumber average molecular weight (Mn) as determined by size exclusionchromatography of at least 1500 g/mol, more specifically 1500 g/mol to1,000,000 g/mol, 4000 g/mol to 150000 g/mol, or 4000 g/mol to 50000g/mol. In an embodiment, the cationic polymer and/or the electrophilicpolymer has a number average molecular weight of 10,000 to 20,000g/mole. The cationic polymer and/or the electrophilic polymer alsopreferably has a narrow polydispersity index (PDI), generally from 1.01to 2.0, more particularly 1.01 to 1.30, and even more particularly 1.01to 1.25.

A cyclic carbonyl monomer comprising a pendant quaternary amine groupcan be used to prepare the cationic polymer. However, these monomers aremore difficult to prepare, are less stable, and the correspondingpolymers tend to be more polydisperse. Therefore, the quaternizationreaction is preferably performed after the ring-openingpolymerization(s).

In aqueous solution, the cationic polymers form unimolecularnanoparticles having an average particle size of 10 nm to 500 nm, 10 nmto 250 nm, and more particularly 50 nm to 200 nm as measured by dynamiclight scattering. For the foregoing particle sizes, the aqueous solutionpreferably has a pH of 4.5 to 8.0, 5.0 to 7.0, or 6.0 to 7.0.

INDUSTRIAL APPLICABILITY

Compositions comprising the cationic polymers have strong antimicrobialproperties against Gram-negative and/or Gram-positive microbes, and havelow cytotoxicity. Furthermore, the cationic polymers can besubstantially or wholly biodegradable, making the compositionsattractive for prevention and treatment of infections caused bydrug-resistant microbes such as methicillin-resistant Staphylococcusaureus (MRSA). Uses include disinfectant washes for hands, skin, hair,bone, ear, eye, nose, throat, internal tissue, wounds, and teeth (e.g.,as a mouthwash).

A method comprises contacting a microbe with the antimicrobialcomposition, thereby killing the microbe.

The cationic polymer can be used as a drug. The drug can be administeredas a powder, a pill, or a liquid solution. The drug can be administeredorally or by way of other body cavities, by injection, intravenously,and/or topically. An injectable composition comprises an aqueous mixtureof the disclosed cationic polymers. A method of treating a cellcomprises contacting the cell with a composition comprising i) thedisclosed cationic polymer and ii) a gene and/or a drug.

The cationic polymers can be applied to human and/or other animaltissues, mammalian and/or non-mammalian tissues. The general term“animal tissue” includes wound tissue, burn tissue, skin, internal organtissue, blood, bones, cartilage, teeth, hair, eyes, nasal surfaces, oralsurfaces, other body cavity surfaces, and any cell membrane surfaces.

The cationic polymers are also attractive as disinfecting agents forsurfaces of articles (i.e., non-living articles) such as, for example,building surfaces in homes, businesses, and particularly hospitals.Exemplary home and commercial building surfaces include floors, doorsurfaces, bed surfaces, air conditioning surfaces, bathroom surfaces,railing surfaces, kitchen surfaces, and wall surfaces.

Also disclosed is an article comprising the disclosed cationic polymerdisposed on a surface of a medical device. Non-limiting medical devicesinclude swabs, catheters, sutures, stents, bedpans, gloves, facialmasks, absorbent pads, absorbent garments, internal absorbent devices,insertable mechanical devices, wound dressings, and surgicalinstruments. Surfaces of the articles can comprise materials such aswood, paper, metal, cloth, plastic, rubber, glass, paint, leather, orcombinations thereof. A method comprises disposing the antimicrobialcomposition on a surface of a medical device, wherein the composition isan effective antimicrobial agent against a Gram-negative microbe andGram-positive microbe.

Loaded Complexes.

The cationic polymer can form a loaded complex (polyplexes) withnegatively charged biologically active substances such as genes,nucleotides, proteins, peptides, drugs, or a combination thereof,thereby providing a therapeutic agent capable of two or more independentbiological functions (e.g., antimicrobial function, gene and/or drugdelivery function, and/or cell recognition function). A method fortreating a cell and/or a surface comprises i) forming a loaded complexcomprising the cationic polymer and a biologically active substancebound by non-covalent interactions; and ii) contacting the cell and/orthe surface with the loaded complex. The cells can be exposed to theloaded complex in vitro, ex vivo and then subsequently placed into ananimal, or in vivo (for example, an animal or human). In an embodiment,the biologically active substance is a gene, the loaded complex enters acell, the gene is released by the loaded complex within the cell, andthe gene is expressed by the cell. In another embodiment, thebiologically active substance is a drug and/or a protein.

Exemplary commercially available drugs include 13-cis-Retinoic Acid,2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU,6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®,Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®,Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®,Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine,BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide,BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225,Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine,Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013,CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil,Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11,Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal,Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®,Deltasone®, Denileukin Diftitox, DepoCyt™, Dexamethasone, DexamethasoneAcetate, Dexamethasone Sodium Phosphate Dexasone, Dexrazoxane, DHAD,DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal,Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™,Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux,Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos®,Etoposide, Etoposide Phosphate, Eulexin®, Everolimus, Evista®,Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec™,Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte—Colony StimulatingFactor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®,Herceptin®, Hexadrol, Hexylen®, Hexamethylmelamine, HMM, Hycamtin®,Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone SodiumPhosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate,Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan Idamycin®, Idarubicin,Ifex®, IFN-alpha Ifosfamide, IL-11 IL-2 Imatinib mesylate, ImidazoleCarboxamide Interferon alfa, Interferon Alfa-2b (PEG Conjugate),Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®,Irinotecan, Isotretinoin, Ixabepilone, Ixempra™, K Kidrolasei (t),Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™,Liposomal Ara-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®,Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, MechlorethamineHydrochloride, Medralone®, Medrol®, Megace®, Megestrol, MegestrolAcetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate,Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin,Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, MustineMutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine,Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®,Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®,Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxa1™,Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, PaclitaxelProtein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®,Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRONT™,PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard,Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a) Rubex®, Rubidomycinhydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza, and Zometa.

Any cell that can be transfected by a non-viral vector can be treatedwith the above-described loaded complexes. In particular the cells canbe eukaryotic cells, mammalian cells, and more particularly rodent orhuman cells. The cells can be derived from various tissues, includingextraembryonic or embryonic stem cells, totipotent or pluripotent,dividing or non-dividing, parenchyma or epithelium, immortalized ortransformed, or the like. The cell can be a stem cell or adifferentiated cell. Cell types that are differentiated includeadipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,dendritic cells, neurons, glia, mast cells, blood cells and leukocytes(e.g., erythrocytes, megakaryotes, lymphocytes, such as B, T and naturalkiller cells, macrophages, neutrophils, eosinophils, basophils,platelets, granulocytes), epithelial cells, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine orexocrine glands, as well as sensory cells.

The above-described loaded complexes can be used as non-viraltransfection vectors. The target gene is not limited to any particulartype of target gene or nucleotide sequence. For example, the target genecan be a cellular gene, an endogenous gene, an oncogene, a transgene, aviral gene, or translated and non-translated RNAs. Exemplary possibletarget genes include: transcription factors and developmental genes(e.g., adhesion molecules, cyclin-dependent kinase inhibitors, Wntfamily members, Pax family members, Winged helix family members, Hoxfamily members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2,CBL, CSFIR, ERBA, ERBB, ERBB2, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS,JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIMI, PML,RET, SKP2, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC,BRAI, BRCA2, CTMP, MADH4, MCC, NF1, NF2, RB1, TP53, and WTI); andenzymes (e.g., ACP desaturases and hydroxylases, ADP-glucosepyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cyclooxygenases, decarboxylases,dextrinases, DNA and RNA polymerases, galactosidases, glucose oxidases,GTPases, helicases, integrases, insulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, peroxidases,phosphatases, phospholipases, phosphorylases, proteinases andpeptidases, recombinases, reverse transcriptases, telomerase, includingRNA and/or protein components, and topoisomerases).

Charge Shifting.

The release of a biologically active substance can be facilitated bycationic polymers capable of charge-shifting. In charge shifting, thenet positive charge of the cationic polymer is reduced by the conversionof a non-charged group on the cationic polymer side chain into anegatively charged group after the loaded complex has entered the cell.A cationic polymer capable of charge-shifting can comprise, for example,a latent carboxylic acid group, such as an acetal ester, in addition tothe quaternary amine. The acetal ester group has the general formula(16):

wherein the starred bond represents the attachment point to a cycliccarbonyl moiety, and R^(c) and R^(d) are monovalent radicalsindependently comprising from 1 to 20 carbons. In an embodiment, R^(c)is methyl and R^(d) is ethyl. In another embodiment, a diluent cycliccarbonyl monomer is MTCOEE:

Acetal esters can be hydrolyzed under the mildly acidic conditions ofthe endosomal environment (about pH 5) to form a carboxylic acid group.In the more basic environment of the cytosol, the carboxylic acid groupsbecome ionized, thereby lowering the net positive charge of the cationicpolymer and allowing the release of the negative charged biologicallyactive substance. Thus, the cationic polymers can be easily modified totune the charge and the buffering strength for a specific biologicallyactive substance.

Another strategy for facilitating endosomal release involvesnon-covalent interactions to stabilize a biologically active substance,for example, using diluent cyclic carbonyl monomers comprising afluorinated tertiary alcohol group. Fluorinated tertiary alcohol groupsare known to bind to phosphates and related structures, but withinteraction energies that are lower than electrostatic interactions, andhence more easily released.

Other functional groups can be used to facilitate the release of thebiologically active substance from the loaded complex, such as secondaryamine groups, citraconic amide groups, ester groups, and imine groups.

The examples below demonstrate that compositions comprising a cationicpolymer have strong antimicrobial activity against Gram-negativemicrobes, such as Esherichia coli, and Gram-positive microbes, such asStaphylococcus aureus, fungi, and yeast.

EXAMPLES

Materials used in the following examples are listed in Table 11.

TABLE 11 SUP- ABBREVIATION DESCRIPTION PLIER Bis-MPA2,2-Bis(hydroxymethyl)propionic acid, Sigma- MW 134.13 Aldrich DBU1,8-Diazabicyclo[5.4.0]undec-7-ene, Sigma- MW 152.24 Aldrich TUN-bis(3,5-Trifluoromethyl)phenyl-N′- Prepared cyclohexylthiourea belowSparteine (−)-sparteine; (6R,8S,10R,12S)-7,15- Sigma-Diazatetracyclo[7.7.1.02,7.010,15]hepta Aldrich decane, accelerator, MW234.38 PBS Phosphate Buffered Saline Invitrogen TSB Tryptic Soy BrothBecton, Dickinson and Co., USA TMEDAN,N,N′,N′-tetramethylethylenediamine, Merck, MW 116.24 Singapore TCTPTissue Culture Plate, Nunc Nunc MicroWell ™ Treated Polystyrene (CatlogNo. 167008) PFC Bis(pentafluorophenyl) carbonate, MW Central 394.12Glass Co., Ltd. CsF Cesium fluoride; catalyst, MW 151.90 Sigma- Aldrichp-Chloromethyl Benzyl Alcohol, MW Sigma- 156.61 Aldrich DMEAN,N-Dimethylethanolamine, MW 89.14 Sigma- Aldrich PBOH Pyrenebutanol, MW274.36 Sigma- Aldrich MEM Minimal Essential Medium (cell cultureInvitrogen medium) (U.S.A) RPMI-1640 Cell culture medium Invitrogen(U.S.A) FBS Fetal Bovine Serum Invitrogen (U.S.A) TMC TrimethyleneCarbonate (1,3-dioxan-2- Sigma- one), MW 102.09 Aldrich

Herein, Mn is the number average molecular weight, Mw is the weightaverage molecular weight, and MW is the molecular weight of onemolecule.

Diethyl ether, triethylamine (TEA), potassium hydroxide (KOH) andN,N,N′,N′-tetramethylethylenediamine (TMEDA) were purchased from Merck,Singapore. Anhydrous dichloromethane (DCM), dimethylformamide (DMF),tetrahydrofuran (THF) and pyridine were purchased from Sigma-Aldrich.2,2-Bis(hydroxymethyl)propionic acid (bis-MPA), benzyl bromide (BnBr),triphosgene and Pd/C (10%) were purchased from Sigma-Aldrich.1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was dried with CaH₂ overnightand distilled under reduced pressure before stored in a glove box.Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) andCandida albicans (C. albicans), Pseudomonas aeruginosa (P. aeruginosa)and Bacillicus subtilis (B. subtilis) were purchased from ATCC. Allother chemicals were of analytical grade, and used as received.

Polymer Characterization.

The nuclear magnetic resonance (¹H-NMR) spectra of the polymers werestudied using a Broker Avance 400 spectrometer (400 MHz), andchloroform-d (CDCl₃) was used as the solvent. The molecular weights andpolydispersity indices were determined by a gel permeationchromatography (GPC) (Waters 2690, MA, USA, mobile phase: THF at 1.0ml/min, relative to polystyrene standards).

I. Monomer Synthesis

Scheme 3 illustrates pathways for preparing cyclic carbonate monomersfrom 2,2-bis(methylol)propionic acid (bis-MPA).

In Scheme 3, (i) 2,2-bis(methylol)propionic acid (bis-MPA) is convertedto a benzyl ester BnMPA (herein also used as an initiator for thepolymerizations). In reaction (ii) BnMPA reacts with triphosgene to forma cyclic carbonate monomer, MTCOBn. MTCOBn is debenzylated in (iii) toproduce the cyclic carbonyl carboxylic acid, MTCOH. Two pathways areshown for forming an ester from MTCOH. In the first pathway, (iv), MTCOHis treated with a suitable carboxy activating agent, such asdicyclohexylcarbodiimide (DCC), which reacts with ROH to form MTCOR in asingle step. Alternatively, MTCOH can be converted first (v) to the acidchloride MTCCl followed by treatment (vi) of MTCCl with ROH in thepresence of a base to form MTCOR. Both pathways are illustrative and arenot meant to be limiting. The following conditions are typical for thereactions of Scheme 3: (i) Benzylbromide (BnBr), KOH, DMF, 100° C., 15hours, 62% yield of the benzyl ester of bisMPA; (ii) triphosgene,pyridine, CH₂Cl₂, −78° C. to 0° C., 95% yield of MTCOBn; (iii) Pd/C(10%), H₂ (3 atm), EtOAc, room temperature, 24 hours, 99% yield ofMTCOH; (iv) ROH, DCC, THF, room temperature, 1 to 24 hours; (v) (COCl)₂,THF, room temperature, 1 hour, 99% yield of MTCCl; (vi) ROH, NEt₃, roomtemperature, 3 hours yields MTCOR.

Cyclic carbonate haloesters MTCOPrBr, MTCOPrCl, MTCOEtI can be preparedby reaction of MTCCl with 3-bromopropanol, 3-choloropropanol, and2-iodoethanol, respectively, as described below for MTCOPrCl. Thehaloesters were purified by either recrystallization or by flashchromatography (ethyl acetate/hexane) in high yields (>85%).

Example 1 Preparation of MTCOPrCl, MW 236.65

A catalytic amount (3 drops) of DMF was added to a THF solution (200 mL)of MTCOH (11.1 g, 69 mmol), followed by a solution of oxalyl chloride(7.3 mL, 87 mmol) in THF (100 mL), gently added over 20 min under N₂atmosphere. The solution was stirred for 1 hour, bubbled with N₂ flow toremove volatiles, and evaporated under vacuum to give the intermediateMTCCl. A mixture of 3-chloro-1-propanol (5.4 mL, 76 mmol) and pyridine(6.2 mL, 65 mmol) in dry THF (50 mL) was added dropwise to a dry THFsolution (100 mL) of the intermediate MTCCl over 30 min, whilemaintaining a solution temperature below 0° C. with an ice/salt bath.The reaction mixture was kept stirring for another 3 hours at roomtemperature before it was filtered and the filtrate evaporated. Theresidue was dissolved in methylene chloride and washed with 1N HClaqueous solution, saturated NaHCO₃ aqueous solution, brine and water,stirred with MgSO₄ overnight, and the solvent evaporated. The crudeproduct was passed through a silica gel column by gradient eluting ofethyl acetate and hexane (50/50 to 80/20) to provide the product as acolorless oil that slowly solidified to a white solid (9.8 g, 60%).

Example 2 Preparation of Ethyl 2,2-bis(methylol)propionate (EtMPA),Molecular Weight 162.2

2,2-Bis(methylol)propionic acid (bis-MPA; MW 134.1, 22.1 g, 0.165 mol)was added in ethanol (150 mL) with Amberlyst-15 (6.8 g) and refluxedovernight. The resins were then filtered out and the filtrate wasevaporated. Methylene chloride (200 mL) was added to the resultingviscous liquid to filtrate the unreacted reagent and byproduct. Afterthe solution was dried over MgSO4 and evaporated, ethyl2,2-bis(methylol)propionate (EtMPA) was obtained as a clear andcolorless liquid (21.1 g, 86%).

Example 3 Preparation ofN-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU)

TU was prepared as reported by R. C. Pratt, B. G. G. Lohmeijer, D. A.Long, P. N. P. Lundberg, A. Dove, H. Li, C. G. Wade, R. M. Waymouth, andJ. L. Hedrick, Macromolecules, 2006, 39 (23), 7863-7871, and dried bystirring in dry THF over CaH₂, filtering, and removing solvent undervacuum.

Example 4 Preparation of MTCOEt, MW 188.2

MTCOEt was prepared from EtMPA as a non-functional counterpart fordilution effects and to introduce hydrophobic repeat units in thepolycarbonate chain.

A solution of triphosgene (19.5 g, 0.065 mol) in CH₂Cl₂ (200 mL) wasadded stepwise to a CH₂Cl₂ solution (150 mL) of ethyl2,2-bis(methylol)propionate (EtMPA) (21.1 g, 0.131 mol) and pyridine (64mL, 0.786 mol) over 30 min at −75° C. with dry ice/acetone. The reactionmixture was kept stirring for another 2 hours under chilled conditionand then allowed to heat to room temperature. Saturated NH₄Cl aqueoussolution (200 mL) was added to the reaction mixture to decompose excesstriphosgene. The organic phase was then treated with 1N HCl aq (200 mL),followed by saturated NaHCO₃ (200 mL), brine (200 mL), and water (200mL). After the CH₂Cl₂ solution was dried over MgSO₄ and evaporated, theresidue was recrystallized from ethyl acetate to give white crystals(13.8 g, 56%). ¹H NMR: delta 4.68 (d, 2H, CH₂OCOO), 4.25 (q, 1H,OCH₂CH₃), 4.19 (d, 2H, CH₂OCOO), 1.32 (s, 3H, CH₃), 1.29 (t, 3H,CH₃CH₂O). ¹³C NMR: delta 171.0, 147.5, 72.9, 62.1, 39.9, 17.3, 13.8.HR-ESI-MS: m/z calcd for C₈H₁₂O₅; Na, 211.0582; found, 221.0578.

An alternative route to functionalized cyclic carbonate monomersutilizes a cyclic carbonate monomer intermediate bearing an activepentafluorophenyl ester, MTC-PhF₅, which is formed in a single step frombis-MPA as follows.

Example 5 Preparation of MTCOC₆F₅, MW 326

A 100 mL round bottom flask was charged with bis-MPA (5.00 g, 37 mmol),bis-(pentafluorophenyl) carbonate (PFC, 31.00 g, 78 mmol), and CsF (2.5g, 16.4 mmol) rinsed in with THF (70 mL). Initially the reaction washeterogeneous. After one hour a clear homogeneous solution was formedthat was allowed to stir for 20 hours. The solvent was removed in vacuoand the residue was re-dissolved in methylene chloride. The solution wasallowed to stand for approximately 10 minutes, at which time thepentafluorophenol byproduct precipitated and could be quantitativelyrecovered. This pentafluorophenol byproduct showed the characteristic 3peaks in the ¹⁹F NMR of pentafluorophenol and a single peak in the GCMSwith a mass of 184. The filtrate was extracted with sodium bicarbonate,water and was dried with MgSO₄. The solvent was evaporated in vacuo andthe product was recrystallized (ethyl acetate/hexane mixture) to giveMTCOC₆F₅ as a white crystalline powder (GCMS single peak with mass of326 g/mol, calculated molecular weight for C₁₂H₇F₅O₅ (326 g/mol)consistent with the assigned structure. ¹H-NMR (400 MHz in CDCl₃): delta4.85 (d, J=10.8 Hz, 2H, CH₂), 4.39 (d, J=10.8 Hz, 2H, CH₂), 1.55 (s, 3H,CH₃).

MTCOC₆F₅ was then used to prepare additional cyclic carbonyl monomers asfollows.

Example 6 Preparation of MTCOBnCl, MW 298.72

A flask was charged with MTCOC₆F₅ (10 g, 30.6 mmol), p-chloromethylbenzyl alcohol (4.8 g, 30.6 mmol), PROTON SPONGE (2 g, 9.3 mmol, atrademark of Sigma-Aldrich) and THF (30 mL). The reaction mixture wasstirred for 12 hr then added directly to a silica gel column. Theproduct was isolated using diethyl ether as the eluent to yield 7.45 g(81%) white crystalline powder.

Example 7 Preparation of MTCOBuOBn, MW 322.35

A flask was charged with MTCOC₆F₅ (10 g, 30.6 mmol),4-benzyloxy-1-butanol (5.5 g, 30.6 mmol), PROTON SPONGE (2 g, 9.3 mmol)and THF (30 mL). The reaction mixture was stirred for 12 hours thenadded directly to a silica gel column. The product was isolated usingdiethyl ether as the eluent to yield 7.84 g (78%) white crystallinepowder.

Example 8 Preparation of Bn-bis-MPA, MW 222.09

This preparation followed the procedure described by Frechet, et al.,JACS (2001), 123, 5908. 2,2-Bis(hydroxymethyl)-propionic acid 20.00 g(149 mmol), 34.04 g (222 mmol) benzaldehyde dimethyl acetal, and 1.42 g(7.4 mmol) p-toluenesulfonic acid monohydrate (TsOH) were mixed in 150mL of acetone. The reaction mixture was stirred for 4 hours at ambienttemperature. After storage of the reaction mixture in the refrigeratorovernight, solids were filtered off and washed with cold acetone to giveprotected Bn-bis-MPA as white crystals: 21.0 g, (64%). IR (cm⁻¹, thinfilm from CHCl₃): 3400-2300 (br), 1699 (s). ¹H NMR (500 MHz, CDCl₃):delta 1.11 (s, 3), 3.70 (d, 2, J) 11.7), 4.63 (d, 2, J) 11.4), 5.49 (s,1), 7.37 (m, 3), 7.48 (m, 2). ¹³C NMR (500 MHz, CDCl₃): delta 17.58,41.58, 72.65, 100.37, 126.10, 128.01, 128.70, 138.39, 175.58. Calcd.:[M]+m/z) 222.24. Found: TOFMS-ES: [M+Na]+) 245.10. Anal. Calcd forC₁₂H₁₄O₄: C, 64.89; H, 6.52.

Example 9 Preparation of Bn-bis-MPA Anhydride, MW 426.17

Bn-bis-MPA (20.00 g, 90 mmol) and 10.21 g (49.5 mmol) ofN,N′-dicyclohexylcarbodiimide (DCC) were mixed in 150 mL of CH₂Cl₂. Thereaction mixture was stirred overnight at room temperature. Theprecipitated urea DCC byproduct was filtered off in a glass filter andwashed with a small volume of CH₂Cl₂. The crude product was purified byprecipitating the filtrate into 1000 mL of hexane under vigorousstirring. After filtration, Bn-bis-MPA anhydride was isolated as whitecrystals: 17.1 g (89%). IR (cm-1, thin film from CHCl₃): 3050, 1814 (s),1746 (s). ¹H NMR (300 MHz, CDCl₃): delta 1.12 (s, 6), 3.69 (d, 4, J)10.5), 4.66 (d, 4, J) 10.5), 5.47 (s, 2), 7.35 (m, 6), 7.45 (m, 4). ¹³CNMR (400 MHz, CDCl₃): delta 16.85, 44.18, 73.17, 102.11, 126.27, 128.22,129.09, 137.56, 169.12. Calcd.: [M]+m/z) 426.46. Found: TOFMS-ES:[M+Na]+) 449.20. Anal. Calcd for C₂₄H₂₆O₇: C, 67.59; H, 6.15.

II. Initiator Synthesis (Polyol) Example 10 Preparation of DendrimerG-1(OH)₈, MW 600.61

The following is representative of the preparation of a dendritic polyolROP initiator (star polymer). A flask was charged with pentaerythritol,Bn-bis-MPA anhydride (1.2 equivalents per hydroxyl group) DMAP, pyridineand THF. The reaction mixture was stirred until all hydroxyl groups wereesterified. Water was then added to hydrolyze any unreacted anhydrideand then the product was washed with Na₂CO₃ followed by 1 M HCl. Thebenzyl protecting groups were removed by hydrogenolyis using Pd/C,EtOAc/MeOH, and H₂ (40 psi).

Example 11 Preparation of Dendrimer G-2(OH)₁₆, MW 1529.53

G-2(OH)₁₆ was prepared from G-1(OH)₈ following the general proceduredescribed above for the preparation of G-1(OH)₈.

Example 12 Preparation of Dendrimer G-3(OH)₃₂, MW 3387.37

G-3(OH)₃₂ was prepared from G-1(OH)₁₆ following the general proceduredescribed above for the preparation of G-1(OH)₈.

Example 13 Preparation of Linear Polycarbonate ROP Initiator PC-1

In a N₂ filled glovebox a flask was charged with MTCOEt (0.65 g, 3.45mmol), MTCOBuOBn (0.69 g, 2.02 mmol), thiourea (TU) (0.05 g, 0.14 mmol),BnMPA (0.0113 g, 0.05 mmol) and DCM (2 mL). Upon complete dissolution(−)-sparteine (0.03 g, 0.13 mmol) was added with stirring to initiatethe polymerization. After 6 hours the reaction mixture was precipitatedinto cold 2-propanol to yield 1.12 g (80.5%) of benzyl protectedamorphous polycarbonate random copolymer. Mn 12.5 kDa; PDI 1.08. Thebenzyl protected polymer was deprotected by hydrogenolysis using Pd/Cand hydrogen. The vertical stacking of the repeat units in the structureof PC-1 indicates a random polymer chain. In the above structure forPC-1, m and n represent average numbers of repeat units in the chain,and m=68 and n=40. PC-1 was not endcapped. The benzyl protected polymerwas then dissolved in EtOAc (40 mL) along with suspended Pd/C (0.1 g,10%). The reaction vessel was then pressurized to 45 atm H₂ and reactedfor 16 hours. The insoluble materials were filtered and all volatilesremoved by evaporation (0.94 g, 82%). The resulting PC-1 polymercontained an average of 42 hydroxyl groups (ROP initiating sites).

III. Ring Opening Polymerizations (ROP) Using Polyol Initiators andElectrophilic Cyclic Carbonate Monomer MTCOBnCl Example 14 Preparationof Electrophilic Star Polymer ES-1 by ROP Initiated with G-1(OH)₈

In the above structure for ES-1, j and k represent average numbers ofrepeat units per chain, and j=20 and k=30. The electrophilic starpolymer ES-1 was synthesized in a N₂ filled glovebox by first dissolvingthe G-1(OH)₈ (0.04 g, 0.065 mmol) in DMSO (0.15 mL). To this solutionwas added TU (0.1 g, 0.5 mmoles) and (−)-sparteine (0.1 g, 0.4 mmoles).Separately, a solution of L-lactide monomer (1.5 g, 10.4 mmol, MW144.13) in DCM (6 mL) was added slowly to the initiator/catalystsolution at 25° C. careful to maintain homogeneity. The reaction mixturewas then precipitated into 2-propanol and dried under vacuum to yield1.5 g (97%) white polymer. The second block was installed by dissolvingthe previous polymer (0.05 g, 0.00052 mmol) in DCM (1 mL) along with TU(0.03 g, 0.08 mmol) and MTCOBnCl monomer (0.15 g, 0.5 mmol) in a vial.The polymerization was initiated by the addition of (−)-sparteine (0.015g, 0.06 mmol). Upon complete conversion acetyl chloride (0.05 g, 0.6mmol) was added and the product polymer was precipitated into cold2-propanol yielding 0.18 g (84%) white amorphous material. Mn=124 kDa;PDI=1.04.

Each of the hydroxy groups in the G-1(OH)₈ structure can act as aninitiating site for ring opening polymerization. Sequential ring openingpolymerization of L-lactide (ROP-1) initiated by G-1(OH)₈ producedIntermediate-1 having a living chain end, which is capable of initiatinga second ring opening polymerization of MTCOBnCl (ROP-2), therebyproducing Intermediate-2, also having a living end unit. Intermediate-2was endcapped using acetic anhydride to form electrophilic star polymerES-1. The brackets subtended by 8 in the structure of ES-1 indicatethere are 8 block copolymer chains (arms) linked to the G′ core. Each ofthe starred bonds in G′ represents an attachment point of a blockcopolymer chain to a residual oxygen of a ROP initiator group ofG-1(OH)₈. The inner block of the block copolymer chain, which is linkeddirectly to the G′ core, is a polyester homopolymer block(poly(L-lactide)) derived from L-lactide. The peripheral block linked tothe poly(L-lactide) block is a polycarbonate homopolymer block derivedfrom MTCOBnCl.

ES-2 and ES-3 were prepared by a ROP polymerization initiated byG-2(OH)₁₆ and G-3(OH)₃₂, respectively, according to the above-describedprocedure for the preparation of ES-1, adjusted for differences in thenumber of initiating sites.

Example 15 Preparation of Electrophilic Precursor Star Polymer ES-2 fromG-2(OH)₁₆

In the above structure for ES-2, j=20 and k=30. ES-2 was synthesizedaccording to the same procedure as ES-1 adjusting only for the increasednumber of polymer arms. G-2(OH)₁₆ (0.05 g, 0.0033 mmol was dissolved inDMSO (0.15 mL). To this solution was added TU (0.1 g, 0.5 mmol) and(−)-sparteine (0.1 g, 0.4 mmol). Separately, a solution of L-lactidemonomer (1.5 g, 10.4 mmol) in dichloromethane (DCM) (6 mL) was addedslowly to the initiator/catalyst solution at 25° C. careful to maintainhomogeneity. The reaction mixture was then precipitated into 2-propanoland dried under vacuum to yield 1.5 g (97%) white polymer. The secondblock was installed by dissolving the previous polymer (0.05 g, 0.00026mmol) in DCM (1 mL) along with TU (0.03 g, 0.08 mmol) and MTCOBnClmonomer (0.15 g, 0.5 mmol) in a vial. The polymerization was initiatedby the addition of (−)-sparteine (0.015 g, 0.06 mmol). Upon completeconversion acetyl chloride (0.05 g, 0.6 mmol) was added and the productpolymer was precipitated into cold 2-propanol yielding 0.18 g (90%)white amorphous material. Mn=174 kDa; PDI=1.04. The brackets subtendedby 16 in the structure of ES-2 indicate there are 16 block copolymerchains (arms) linked to the G″ core.

Example 16 Preparation of Electrophilic Precursor Star Polymer ES-3 fromG-3(OH)₃₂

In the above structure for ES-3, j=20 and k=30. ES-3 was synthesizedaccording to the same procedure as ES-1 adjusting only for the increasednumber of polymer arms. G-3(OH)₃₂ (0.056 g, 0.0016 mmol) was dissolvedin DMSO (0.15 mL). To this solution was added TU (0.1 g, 0.5 mmol) and(−)-sparteine (0.1 g, 0.4 mmol). Separately, a solution of L-lactidemonomer (1.5 g, 10.4 mmol) in DCM (6 mL) was added slowly to theinitiator/catalyst solution at 25° C. careful to maintain homogeneity.The reaction mixture was then precipitated into 2-propanol and driedunder vacuum to yield 1.4 g (90%) white polymer. The second block wasinstalled by dissolving the previous polymer (0.05 g, 0.00013 mmol) inDCM (1 mL) along with TU (0.03 g, 0.08 mmol) and MTCOBnCl monomer (0.15g, 0.5 mmol) in a vial. The polymerization was initiated by the additionof (−)-sparteine (0.015 g, 0.06 mmol). Upon complete conversion acetylchloride (0.05 g, 0.6 mmol) was added and the product polymer wasprecipitated into cold 2-propanol yielding 0.17 g (85%) white amorphousmaterial. Mn=174 kDa; PDI=1.04. The brackets subtended by 32 in thestructure of ES-3 indicate there are 32 block copolymer chains (arms)linked to the G′″ core.

Example 17 Preparation of Electrophilic Graft Polymer EG-1

In the above structure for EG-1, w=42 (the average number of ROPinitiator sites per PC-1 molecule), m=68, n=40, j=9 and k=28 in theabove structure. The brackets subtended by w in the structure of EG-1indicate there are w=42 block copolymer chains (arms) independentlylinked to core structure A′. The initiator PC-1 was not endcapped. Thatis, the terminal hydroxy groups of PC-1 are capable of initiating a ringopening polymerization, in addition to each side chain hydroxy group.Consequently, in EG-1 and other structures described below w is notnecessarily equal to n in PC-1. As shown, two sequential ring openingpolymerizations were conducted using L-lactide (ROP-1) and MTCOBnCl(ROP-2). The starred bonds in core structure A′ represents attachmentpoints for the block copolymer chain. In this instance, the inner blockdirectly linked to A′ group is a poly(L-lactide) homopolymer block. Theperipheral block is a polycarbonate homopolymer block derived fromMTCOBnCl. EG-1 was not endcapped.

The preparation of EG-1 was as follows. PC-1 (0.05 g, 0.004 mmol),L-lactide (0.12 g, 0.83 mmol), TU (0.03 g, 0.07 mmol) and DCM (0.7 mL)were added to a vial. (−)-Sparteine (0.015 g, 0.06 mmol) was then addedto initiate the polymerization. Upon full conversion, the reactionmixture was precipitated into 2-propanol and dried yielding 0.15 g (89%)white polymer. A portion of this material (0.05 g, 0.0007 mmol) wascombined with MTCOBnCl (0.27 g, 0.9 mmol), TU (0.04 g, 0.1 mmol) and DCM(0.6 mL). The polymerization was then initiated by adding (−)-sparteine(0.04 g, 0.17 mmol). Upon full conversion the reaction mixture wasprecipitated into 2-propanol yielding 0.29 g (90%).

The following preparation of EG-2 utilized PC-1 to initiate a single ROPpolymerization of cyclic carbonate monomer MTCOBnCl. The resultingelectrophilic graft polymer EG-2 comprises a pendant polycarbonatehomopolymer chain directly linked to core structure A′.

Example 18 Preparation of Electrophilic Graft Polymer EG-2 Having CoreStructure A′ Shown Above

In the structure above for EG-2, m=68, n=40, k=9, and w=42 (the averagenumber of ROP initiating sites on PC-1). The brackets subtended by w inthe structure of EG-2 indicate there are w=42 polycarbonate chains(arms) independently linked to core structure A′ (see Example 17). Thestarred bonds in core structure A′ represents attachment points for thepolycarbonate chains. EG-2 was prepared as follows. A vial was chargedwith PC-1 (0.05 g, 0.004 mmol), MTCOBnCl (0.26 g, 0.83 mmol), TU (0.04g, 0.1 mmol) and DCM (0.6 mL). The polymerization was then initiatedwith the addition of sparteine (0.4 g, 0.17 mmol). Upon full conversionthe reaction mixture was precipitated into 2-propanol yielding a whitepolymer, EG-2.

The following preparation of electrophilic graft polymer EG-3 utilizedPC-1 to initiate a single ROP polymerization of cyclic carbonate monomerMTCOPrCl. As in EG-2, EG-3 comprises a pendant polycarbonate homopolymerchain directly linked to core structure A′.

Example 19 Preparation of Electrophilic Graft Polymer EG-3 Having CoreStructure A′

In the structure above for EG-3, m=68, n=40, k=9, and w=42 (the averagenumber of ROP initiating sites in PC-1). EG-3 was prepared as follows.The brackets subtended by w in the structure of EG-3 indicate there arew=42 polycarbonate chains (arms) independently linked to core structureA′ (see Example 17). The starred bonds in core structure A′ representsattachment points for the polycarbonate chains. The polymer wassynthesized by charging a vial with PC-1 (0.05 g, 0.004 mmol), MTCOPrCl(0.20 g, 0.83 mmol), TU (0.04 g, 0.1 mmol) and DCM (0.6 mL). Thepolymerization was then initiated with the addition of sparteine (0.4 g,0.17 mmol). Upon full conversion the reaction mixture was precipitatedinto 2-propanol yielding a white polymer, EG-3.

The following electrophilic linear polycarbonate copolymer, EL-1, wasformed by ROP polymerization of a mixture of trimethylene carbonate(TMC) and MTCOBnCl initiated by pyrene butanol (PBOH).

Example 20 Preparation of Electrophilic Linear Random PolycarbonateCopolymer EL-1

In the structure of EL-1, j=30, and k=10. EL-1 was prepared as follows:PBOH (0.045 g, 0.163 mmol), TMC (0.5 g, 4.9 mmol), MTCOBnCl (0.487 g,1.63 mmol), TU (0.06 g, 0.163 mmol) and DCM (3 mL) were added to a vial.The polymerization was then initiated by adding (−)-sparteine (0.04 g,0.163 mmol). Upon full conversion the reaction mixture was precipitatedinto MeOH yielding 0.93 g (90%).

Example 21 Preparation of Electrophilic Graft Polymer EG-4 Having CoreStructure A′

In the above structure of EG-4, w=42 (the average number of ROPinitiating sites in PC-1), m=68, n=40, j=9, and k=14. A′ has thestructure shown in Example 17. EG-4 was prepared by the procedure usedin EG-1, substituting L-lactide with trimethylene carbonate (TMC). Avial was charged with PC-1 (0.05 g, 0.004 mmol), TMC (0.084 g, 0.83mmol), TU (0.04 g, 0.1 mmol) and DCM (0.6 mL). The polymerization wasthen initiated with the addition of (−)-sparteine (0.4 g, 0.17 mmol).Upon full conversion the reaction mixture was precipitated into2-propanol yielding a white polymer. This polymer was redissolved in DCM(1 mL) along with MTCOBnCl (0.747 g, 2.49 mmol) and TU (0.04 g, 0.1mmol). The polymerization was then initiated by adding (−)-sparteine(0.45 g, 0.19 mmol). Upon full conversion the reaction mixture wasprecipitated into 2-propanol yielding 0.68 g (77%) white polymer.

The electrophilic polymers are summarized in Table 12.

TABLE 12 Electrophilic ROP-1 ROP-2 j k Polymer Core Type InitiatorMonomer(s) Monomer (mol %) (mol %) ES-1 G′ star G-1(OH)₈ L-LactideMTCOBnCl 20 30 (40%) (60%) ES-2 G″ star G-2(OH)₁₆ L-Lactide MTCOBnCl 2030 (40%) (60%) ES-3 G″ star G-3(OH)₃₂ L-Lactide MTCOBnCl 20 30 (40%)(60%) EG-1 A′ graft PC-1 L-Lactide MTCOBnCl  9 28 (24.3%)   (76.7%)  EG-2 A′ graft PC-1 MTCOBnCl  0  9 (100%)  EG-3 A′ graft PC-1 MTCOPrCl  09 (100%)  EG-4 A′ graft PC-1 TMC MTCOBnCl  9 14 (40%) (60%) EL-1 nonelinear PBOH TMC/ 30 10 MTCOBnCl (75%) (25%) (TMC) MTCOBnCl

IV. Quaternization Reactions

General procedure for quaternization with trimethylamine (TMA). Theelectrophilic polymer was added to a vial and dissolved in acetonitrile.The vial was then cooled to −78° C. and TMA gas was added. The vial wasthen warmed to room temperature and allowed to stir overnight. Thereaction mixture was then precipitated into diethyl ether.

General procedure for quaternization withN,N,N′,N′-tetramethylethylenediamine (TMEDA) andN,N-dimethylethanolamine (DMEA). Electrophilic polymer was added to avial and dissolved in MeCN. To the vial was added TMEDA. The vial wasthen allowed to stir overnight at room temperature. The reaction mixturewas precipitated into diethyl ether.

General procedure for quaternization with N,N-dimethylethanolamine(DMEA). Electrophilic polymer was added to a vial and dissolved in MeCN.To the vial was added DMEA. The vial was then allowed to stir overnightat room temperature. The reaction mixture was precipitated into diethylether.

In the following structures, G′, G″, and G″′ have the structures shownin Examples 14, 15, and 16, respectively. A′ has the structure shown inExample 17.

Example 22 Preparation of Cationic Star Polymer CS-1 Having CoreStructure G′

Following the general procedure described above ES-1 was quaternizedusing TMA, resulting in cationic star polymer CS-1 shown above. In theabove structure of CS-1, j=20 and k=30.

Example 23 Preparation of Cationic Star Polymer CS-2 Having CoreStructure G″

Following the general quaternization procedure described above,electrophilic star polymer ES-2 was quaternized using TMA to producecationic star polymer CS-2 shown above. In the above structure of CS-2,j=20 and k=30.

Example 24 Preparation of Cationic Star Polymer CS-3 Having CoreStructure G″′

Following the general procedure described above, electrophilic starpolymer ES-3 was quaternized using TMA to produce cationic star polymerCS-3 shown above. In the above structure of CS-3, j=20 and k=30.

Example 25 Preparation of Cationic Graft Polymer CG-1 Having CoreStructure A′

Following the general procedure described above, electrophilic graftpolymer EG-1 was quaternized using TMA to form cationic graft polymerCG-1 shown above. In the above structure of CG-1, w=42, j=10, k=30. InA′ of CG-1, m=68 and n=40.

Example 26 Preparation of Cationic Graft Polymer CG-2 Having CoreStructure A′

Following the general procedure described above, electrophilic graftpolymer EG-2 was quaternized using TMA, resulting in cationic graftpolymer CG-2 shown above. In the above structure of CG-2, w=42, andk=10. In A′ of CG-2, m=68 and n=40.

Example 27 (Comparative) Preparation of Cationic Graft Polymer CG-3Having Core Structure A′

Following the general procedure described above, electrophilic graftpolymer EG-3 was quaternized using TMA, resulting in cationic graftpolymer CG-3 shown above. In the above structure of CG-3, w=42 and k=10.In A′ of CG-3, m=68 and n=40.

Example 28 (Comparative) Preparation of Cationic Linear Random CopolymerCL-1

Following the general procedure described above, electrophilic linearrandom polymer EL-1 was quaternized using TMA to form cationic linearrandom copolymer CL-1. In the above structure of CL-1, j=28.5 (3100 kDa)and k=6.8 (2340 kDa).

Example 29 Preparation of Cationic Star Polymer CS-4 Having CoreStructure G′

Following the general procedure described above, ES-1 was quaternizedusing TMEDA, resulting in cationic star polymer CS-4 shown above. In theabove structure of CS-4, j=20 and k=30.

Example 30 Preparation of Cationic Star Polymer CS-5 Having CoreStructure G″

Following the general procedure described above, electrophilic starpolymer ES-2 was quaternized using TMEDA, resulting in cationic starpolymer CS-5 shown above. In the above structure of CS-5, j=20 and k=30.

Example 31 Preparation of Cationic Star Polymer CS-6 Having CoreStructure G′″

Following the general procedure described above, electrophilic starpolymer ES-3 was quaternized using TMEDA, resulting in cationic starpolymer CS-6 shown above. In the above structure of CS-6, j=20 and k=30.

Example 32 Preparation of Cationic Star Polymer CS-7 Having CoreStructure G″′ Shown Above

The procedure for Example 10 was repeated using N,N-dimethylethanolaminefor the quaternization, resulting in cationic star polymer CS-7 shownabove. In the above structure of CS-7, j=20 and k=30.

Example 33 Preparation of Cationic Graft Polymer CG-4 Having CoreStructure A′

Electrophilic graft polymer EG-4 was quaternized using TMA using thegeneral procedure described above to prepare cationic graft polymer CG-4shown above. In the above structure of CG-4, w=42, j=10, and k=30. In A′of CG-4, m=68 and n=40.

Example 34 Preparation of Cationic Graft Polymer CG-5 Having CoreStructure A′ Shown Above

Using the general procedure described above, electrophilic graft polymerEG-1 was quaternized using TMEDA to form cationic graft polymer CG-5shown above. In the above structure of CG-5, w=42, j=10 and k=30. In A′of CG-5, m=68 and n=40.

Example 35 Preparation of Cationic Graft Polymer CG-6 Having CoreStructure A′ Shown Above

Using the general procedure described above, electrophilic graft polymerEG-4 was quaternized using TMEDA to form cationic graft polymer CG-6shown above. In the above structure of CG-6, w=42, j=10, and k=30. In A′of CG-6, m=68 and n=40.

Example 36 Preparation of Cationic Graft Polymer CG-7 Having CoreStructure A′ Shown Above

Using the general procedure described above, electrophilic graft polymerEG-2 was quaternized using TMEDA to form cationic graft polymer CG-7shown above. In the above structure of CG-7, w=42 and k=30. In A′ ofCG-7, m=68 and n=40.

The cationic polymers are summarized in Table 13. In each Example exceptComparative Example 27 the quaternary nitrogen is directly covalentlylinked to a methylene group of a side chain benzyl moiety. That is, thequaternary nitrogen is indirectly covalently linked to the polymerbackbone through the side chain benzyl group.

TABLE 13 Cationic ROP-1 ROP-2 Example Polymer Core Type InitiatorMonomer Monomer Amine 22 CS-1 G′ star G-1(OH)₈ L-Lactide MTCOBnCl TMA 23CS-2 G″ star G-2(OH)₁₆ L-Lactide MTCOBnCl TMA 24 CS-3 G′″ star G-3(OH)₃₂L-Lactide MTCOBnCl TMA 25 CG-1 A′ graft PC-1 L-Lactide MTCOBnCl TMA 26CG-2 A′ graft PC-1 MTCOBnCl TMA 27(comp.) CG-3 A′ graft PC-1 MTCOPrClTMA 28 CL-1 linear PBOH TMC/ TMA MTCOBnCl 29 CS-4 G′ star G-1(OH)₈L-Lactide MTCOBnCl TMEDA 30 CS-5 G″ star G-2(OH)₁₆ L-Lactide MTCOBnClTMEDA 31 CS-6 G′″ star G-3(OH)₃₂ L-Lactide MTCOBnCl TMEDA 32 CS-7 G′″star G-3(OH)₃₂ L-Lactide MTCOBnCl DMEA 33 CG-4 A′ graft PC-1 TMCMTCOBnCl TMA 34 CG-5 A′ graft PC-1 L-Lactide MTCOBnCl TMEDA 35 CG-6 A′graft PC-1 TMC MTCOBnCl TMEDA 36 CG-7 A′ graft PC-1 MTCOBnCl TMEDA

V. Minimal Inhibitory Concentration (MIC) Measurements

Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Bacillussubtilis, and Pseudomonas aeruginosa (P. aeruginosa) obtained from ATCCwere re-constituted from the lyophilized form according to themanufacturer's protocol. Bacterial samples were cultured in Tryptic SoyBroth (TSB) solution at 37° C. under constant shaking of 100 rpm. TheMICs of the polymers were measured using the broth microdilution methodas reported previously. 100 microliters of TSB broth containing apolymer at various concentrations was placed into each well of a 96-welltissue culture plate (TCTP). An equal volume of bacterial suspension(3×10⁶ CFU/mL) was added into each well. Prior to mixing, the bacterialsample was first inoculated overnight to enter its log growth phase. Theconcentration of bacterial solution was adjusted to give an initialoptical density (O.D.) reading of approximately 0.07 at 600 nm on amicroplate reader (TECAN, Switzerland), which corresponds to a cellconcentration of 3×10⁸ CFU/mL in accordance with McFarland turbiditystandard 1 solution. The bacterial solution was further diluted by100-fold to achieve an initial loading of 3×10⁶ CFU/mL. The 96-wellplate was kept in an incubator at 37° C. under constant shaking of 100rpm for 18 hours. The MIC was taken as the concentration of theantimicrobial polymer at which no microbial growth was observed withunaided eyes and microplate reader (TECAN, Switzerland) at the end of 18hours incubation. Broth containing microbial cells alone was used asnegative control, and each test was carried out in 4 replicates.

MIC data is summarized in Table 14 in units of parts per million (ppm).A lower value indicates greater antimicrobial activity. The term“(comp.)” refers to a comparative example.

TABLE 14 MIC^(a) MIC^(a) MIC^(a) MIC^(a) MIC^(a) Cationic ROP-1 ROP-2(B. (S. (E. (C. (P. Ex. Polymer Initiator Monomer Monomer Aminesubtilis)^(b) aureus)^(b) coli)^(c) albicans)^(b) aeruginosa)^(b) 22CS-1 G-1(OH)₈ L-Lactide MTCOBnCl TMA 500 23 CS-2 G-2(OH)₁₆ L-LactideMTCOBnCl TMA 250 24 CS-3 G-3(OH)₃₂ L-Lactide MTCOBnCl TMA 125 125 25CG-1 PC-1 L-Lactide MTCOBnCl TMA 125 500 63 >1000 26 CG-2 PC-1 MTCOBnClTMA 250 125 63 500 27 CG-3 PC-1 MTCOPrCl TMA >1000 >1000 1000 1000(comp.) 28 CL-1 PBOH TMC/ TMA 500 >500 (comp.) MTCOBnCl (8 hr) (8 hr) 29CS-4 G-1(OH)₈ L-Lactide MTCOBnCl TMEDA 250 30 CS-5 G-2(OH)₁₆ L-LactideMTCOBnCl TMEDA >1000 31 CS-6 G-3(OH)₃₂ L-Lactide MTCOBnCl TMEDA 500 50032 CS-7 G-3(OH)₃₂ L-Lactide MTCOBnCl DMEA >1000 >1000 33 CG-4 PC-1 TMCMTCOBnCl TMA 1000 >1000 63 >1000 34 CG-5 PC-1 L-Lactide MTCOBnCl TMEDA125 500 63 >1000 35 CG-6 PC-1 TMC MTCOBnCl TMEDA 250 >1000 63 >1000 36CG-7 PC-1 MTCOBnCl TMEDA 250 125 31 500 ^(a)Minimum inhibitoryconcentration in mg/L: the concentration at which the growth of bacteriais completely inhibited. ^(b)Gram-positive. ^(c)Gram-negative.

The data in Table 14 demonstrate a significant improvement in theantimicrobial activity (indicated by lower MIC values) when thequaternary amine group has benzyl substituent (compare Example 26 (CG-2)with Comparative Example 27 (CG-3)). Further, both graft and starcationic polymers having a benzyl substituted quaternary amine hadhigher activity against S. aureus compared to a linear polymer having abenzyl substituted quaternary amine (compare Examples 24 (CS-3), 26(CG-2) and 36 (CG-7) with comparative Example 28 (CL-1).

Cationic star polymer Example 24 (CS-3), and cationic graft polymerExamples 26 (CG-2) and 36 (CG-7) prepared from TMA, TMA and TMEDA,respectively, had the strongest antimicrobial activity against S. aureus(MIC 125 mg/L to 250 mg/L) and against E. coli (MIC 125 mg/L to 250mg/L).

Comparing the mol % values for j and k in Table 12 with the MIC data inTable 14, chains P′ can comprise the cationic repeat unit having abenzyl substituted quaternary amine group in an amount of about 60 mol %to 100 mol %, more preferably 70 mol % to 100 mol %, based on totalmoles of repeat units in chains P′.

Comparative Examples 37-39

Cationic linear polymers CL-2, CL-3 and CL-4 were prepared according tothe described procedures in “ANTIMICROBIAL POLYMERS AND METHODS OFMANUFACTURE THEREOF”, published US patent application 2011/0150977 A1 toJ. Hedrick, et al., and tested against Gram-negative E. coli.

Comparative Example 37 CL-2. a=60

Comparative Example 38 CL-3. a=40, b=60

Comparative Example 39 CL-4. Mn=11000; a=2.1, b=10.3, n=10

Comparative Example 40 Preparation of CL-5. m=17 (2850 Da), n=3.5 (1190Da)

A vial was charged with BnMPA initiator (0.036 g, 0.163 mmol), TMC (0.5g, 4.9 mmol), MTCOPrBr (0.46 g, 1.63 mmol), TU (0.121 g, 0.32 mmol), DCM(3.0 g) and a stir bar. The polymerization was initiated by the additionof (−)-sparteine (0.077 g, 0.032 mmol) and stirred at ambienttemperature. Upon complete monomer conversion the polymer product wasendcapped with acetyl chloride and precipitated into cold 2-propanolyielding 0.80 g amorphous polymer. The endcapped intermediate polymerwas treated with trimethylamine in acetonitrile followed by stirring at50° C. for 16 hours to produce CL-5.

Example 41 Preparation of5-methyl-5-(3-bromopropyl)oxycarboxyl-1,3-dioxan-2-one, (MTCOPrBr), MW281.10

MTCOPrBr was prepared by the procedure of Example 1 on a 45 mmol scaleusing 3-bromo-1-propanol as the alcohol. The product was purified bycolumn chromatography, and subsequently recrystallized to yield whitecrystals (6.3 g, 49%). ¹H NMR (400 MHz, CDCl₃): delta 4.69 (d, 2H;CH₂OCOO), 4.37 (t, 2H; OCH₂), 4.21 (d, 2H; CH₂OCOO), 3.45 (t, 2H;CH₂Br), 2.23 (m, 2H; CH₂), 1.33 (s, 3H; CH₃). ¹³C NMR (100 MHz, CDCl₃):delta 171.0, 147.3, 72.9, 63.9, 40.2, 31.0, 28.9, 17.3.

Example 42 Preparation of5-methyl-5-(2-iodoethyl)oxycarboxyl-1,3-dioxan-2-one, (MTCOEtI), MW314.08

MTCOEtI was prepared by the procedure of Example 1 on a 45 mmol scale,using 2-iodoethanol as the alcohol, and was purified by columnchromatography and subsequent recrystallization to yield yellowishcrystals (7.7 g, 54%). ¹H NMR (400 MHz, CDCl₃): delta 4.73 (d, 2H;CH₂OCOO), 4.45 (t, 2H; OCH₂), 4.22 (d, 2H; CH₂OCOO), 3.34 (t, 2H; CH₂I),1.38 (s, 3H; CH₃). ¹³C NMR (100 MHz, CDCl₃): delta 170.5, 147.3, 72.8,65.6, 40.3, 17.5, −0.3.

The MIC data for Comparative Examples 37-39 against E. coli aresummarized in Table 15. Table 15 also includes MIC data againstGram-positive Bacillus subtilis (B. subtilis) previously reported forComparative Examples 37-39 (see Table 10, Examples 20, 22, and 24 inpublished U.S. patent application 2011/0150977 A1 to J. Hedrick, etal.). A lower value indicates greater antimicrobial activity.Comparative Example 40, CL-5, was tested only against S. aureus and B.subtilis.

TABLE 15 MIC^(a) MIC^(a) MIC^(a) (B. subtilis ^(b)) (S. aureus ^(b)) (E.coli ^(c)) Ex. Cationic Polymer (mg/L) (mg/L) (mg/L) 37 CL-262.5^(d) >500 (comp.) 38 CL-3 31.3^(d) >500 (comp.) 39 CL-4 62.5^(d)~500 (comp.) 40 CL-5 250 >500 (comp.) ^(a)Minimum inhibitoryconcentration: a concentration, at which the growth of bacteria iscompletely inhibited. ^(b)Gram-positive. ^(c)Gram-negative. ^(d)From US2011/0150977 A1.

As seen in Table 15, Comparative Examples 37-40 were active againstGram-positive B. subtilis, and only weakly active or not active againstGram-negative E. coli. A concentration of about 500 mg/L or more wasneeded to inhibit growth of E. coli.

VI. Hemolytic Activity Assay

The undesired biological activity of the polymers against mammaliancells was tested using freshly drawn rat red blood cells (rRBCs)obtained from AHU, BRC, Singapore. Thus, rRBCs were subjected to 25×volumetric dilutions in phosphate buffered saline (PBS) to achieve 4%blood content (by volume) as reported previously. Antimicrobialsolutions were prepared by dissolving polymers in PBS at concentrationsranging from 0 mg/L to 2000 mg/L. Equal volumes of antimicrobialsolutions (100 microliters) were then mixed with the diluted bloodsuspension (100 microliters). The mixtures were then incubated at 37° C.for 1 hour to allow for the interactions between rRBC and the polymersto take place. Following the incubation, the mixture was subjected tocentrifugation (3000 g for 5 minutes), after which, the supernatant (100mL) was transferred into a 96-well microplate. The hemoglobin releasewas measured spectrophotometrically by measuring the absorbance of thesamples at 576 nm using a microplate reader (TECAN, Switzerland). Twocontrol groups were provided for this assay: untreated rRBC suspension(as negative control), and rRBC suspension treated with 0.1% Triton-X(as positive control). Each assay was performed in 4 replicates andrepeated 3 times to ensure reproducibility of the experiments.Percentage of hemolysis was as follows: Hemolysis (%)=[(O.D_(576 nm) ofthe treated sample−O.D_(576 nm) of the negative control)/(O.D_(576 nm)of positive control−O.D_(576 nm) of negative control)]×100%, whereO.D_(576 nm) means optical density at 576 nm.

FIG. 1 is a graph of the % hemolysis as a function of concentration inppm of cationic star copolymers CS-3 (Example 24, TMA), CS-6 (Example31, TMEDA) and CS-7 (Example 32, DMEA), each having a G″′ core anddiffering only in the amine used for the quaternization. The % hemolysiswas about 25% using TMA, about 12% using TMEDA, and less than 1% usingDMEA at a concentration of 250 ppm. This indicates that the hydroxylgroup present in DMEA significantly reduced hemolysis.

FIG. 2 is a graph of the % hemolysis as a function of concentration inppm of cationic graft copolymer CG-1 (Example 25), CG-2 (Example 26),CG-3 (comparative Example 27,) and CG-4 (Example 33), each quaternizedwith TMA. In the range of 125 ppm to 250 ppm suitable for antimicrobialuse, the % Hemolysis was less than 10% for each cationic polymer. Theresults demonstrated that the cationic graft polymers had excellentselectivity towards microbes over mammalian cells, and were thereforepromising antimicrobials with broad-spectrum activities.

The % hemolysis using cationic linear polymers CL-1 (Example 28) andCL-5 (Example 40) was less than 10% for cationic polymer concentrationsin a range of 50 mg/L to 500 mg/L.

VII. Gene Delivery and DNA Expression

Plasmid DNA encoding the 6.4 kb firefly luciferase gene driven by thecytomegalovirus (CMV) promoter was obtained from Carl Wheeler, Vical(U.S.A.), amplified in Escherichia coli DH5α and purified using EndofreeGiga plasmid purification kit from Qiagen.

Preparation of DNA complexes with star and graft cationic polycarbonatesand control polymer polyethyleneimine (PEI). The polycarbonate polymeror PEI was first dissolved in 10 mM phosphate buffer (pH 6.0) and HPLCgrade water. To form the DNA complex, an equivolume solution of DNA wasadded dropwise to the polycarbonate polymer or PEI solution to achievethe intended N/P ratio (molar ratio of ammonium (N) content in thepolymer to phosphorous (P) content in the DNA) under gentle vortexingfor approximately 10 seconds. The mixture was equilibrated at roomtemperature for 30 min to allow for complete electrostatic interactionbetween the DNA molecule and polymer (PEI, star or graft cationicpolycarbonate), before being used for subsequent studies.

Gel retardation assay. Various formulations of polymer/DNA complexeswere prepared with various N/P ratios. Post-equilibration, the complexeswere electrophoresed on 0.7% agarose gel (stained with 5 microliters of10 mg/mL ethidium bromide per 50 mL of agarose solution) in 0.5×TBEbuffer at 80V for 50 min. The gel was then analyzed under an UVilluminator (Chemi Genius, Evolve, Singapore) to reveal the relativeposition of the complexed DNA to the naked DNA.

The cationic star polymer CS-6 (Example 31) having 32 arms efficientlycondensed DNA (FIG. 3, a black and white photograph of the UVilluminated agarose gel plate after electrophoresis). The completeretardation of DNA mobility in the gel electrophoresis assay wasachieved at N/P ratio of 4, where N/P ratio is the molar ratio ofnitrogen content in the CS-6 polymer to phosphorus in the DNA.

The cationic graft polymers polyalcohol polymers, CG-5 (Example 34),CG-6 (Example 35), and CG-7 (Example 36) also efficiently condensed DNA(FIG. 4, a black and white photograph of the UV illuminated agarose gelplate after electrophoresis). The complete retardation of DNA mobilityin the gel electrophoresis assay was achieved at N/P ratios of 3, 3 and2, respectively.

In vitro gene expression. HepG2 cells were maintained in MEM growthmedium, while 4T1 cells were cultured in RPMI-1640 growth medium. Allgrowth media used were supplemented with 10% FBS, 1 mM sodium pyruvate,100 U/mL penicillin and 100 mg/mL streptomycin and cultured at 37° C.,under an atmosphere of 5% CO₂ and 95% humidified air. The reporter geneused was the 6.4 kb firefly luciferase gene driven by thecytomegalovirus (CMV) promoter (Carl Wheeler, Vical, U.S.A.). The invitro gene transfection efficiency of the cationic polymer or PEI/DNAcomplexes was investigated using HepG2 and 4T1 cell lines. Cells wereseeded onto 24-well plates at a density of 8×10⁴ and 5×10⁴ cells perwell respectively. After 24 hours, the plating media was replaced with0.5 mL of fresh growth media (containing 10% FBS), followed by theaddition of 50 microliters of complex solution (containing 2.5micrograms DNA). Following 4 hours of incubation, free complexes wereremoved by replacing the media in each well. After a further 68 hours ofincubation, the cell culture media in each well was removed and thecells rinsed once with 0.5 mL of PBS before 0.2 mL of reporter lysisbuffer (purchased from Promega (U.S.A.)) was added to each well. Thecell lysate collected after two cycles of freezing (−80° C., 30 min) andthawing was cleared by centrifugation at 14,000 rpm for 5 min, afterwhich, 20 microliters of supernatant was mixed with 100 microliters ofluciferase substrate for the determination of relative light units (RLU)using a luminometer (Lumat LB9507, Berthold, Germany). The RLU readingswere normalized against the protein concentration of the supernatantdetermined using the BCA protein assay to give the overall luciferaseexpression efficiency. In all in vitro gene expression experiments,naked DNA was used as a negative control and PEI/DNA complexes preparedat the optimal N/P ratio (i.e., 10) were used as the positive control.At N/P=10, PEI induced high gene expression efficiency and providedclose to or more than 50% cell viability. Data are expressed as meanstandard deviations of four replicates.

High luciferase expression efficiency was achieved with cationic starpolymer CS-6 (Example 31) at N/P 40 in HepG2 and 4T1 cell lines (FIG. 5,bar chart). The efficiency of CS-6 was comparable to the efficiency ofPEI at its optimal N/P 10, the standard for in vitro gene expression.Cationic star polymer CS-6 was more efficient in gene transfection thanpreviously reported linear polycarbonates (Z. Y. Ong, et al., Journal ofControlled Release, 152 (2011), pg 120-126).

Cationic graft polymers CG-5 (Example 34), CG-6 (Example 35) and CG-7(Example 36) also induced high luciferase expressionat N/P ratios thatgave rise to more than 80-90% cell viability (i.e., N/P 50, 20 and 20for CG-5, CG-6 and CG-7, respectively) (FIG. 6, bar chart). However, theluciferase expression levels yielded by DNA complexes with CG-5, CG-6and CG-7 at the optimal N/P ratios 50, 20 and 20, respectively) wasstill 5 times lower than that mediated by PEI/DNA complexes. Thecationic graft polymers also mediated lower gene expression efficiencycompared to the cationic star polymer CS-6 (Example 31).

VIII. Cytotoxicity of Cationic Polymers Formed with TMEDA

The cytotoxicity of the cationic polymer/DNA complexes was studied usingthe standard MTT assay protocol. HepG2 and 4T1 cells were seeded onto96-well plates at densities of 10000 and 6000 cells per wellrespectively and allowed to grow to 60-70% confluency before treatment.Cationic polymer/DNA complexes at various N/P ratios were prepared in 10mM phosphate buffer (pH 6.0) as described above. The cells in each wellwere then incubated with sample-containing growth media comprising of 10microliters of polymer/DNA complexes and 100 microliters fresh media for4 hours at 37° C. Following incubation, the wells were replaced withfresh growth media and incubated further for 68 hours. Subsequently, 100microliters of growth media and 10 microliters of MTT solution (5 mg/mLin PBS) were then added to each well and the cells were incubated for 4hours at 37° C. according to the manufacturer's directions. Resultantformazan crystals formed in each well were solubilized using 150microliters of DMSO upon removal of growth media. A 100 microliteraliquot from each well was then transferred to a new 96-well plate fordetermination of absorbance using a microplate spectrophotometer atwavelengths of 550 nm and 690 nm. Relative cell viability was expressedas [(A_(550nm)−A_(690nm))Sample/((A_(550nm)−A_(690nm))Control]×100%,where (A_(550nm)−A_(690nm))Sample is the Sample absorbance at 550 nmminus the Sample absorbance at 690 nm, and (A_(550nm)−A_(690nm))Controlis the Control absorbance at 550 nm minus the Control absorbance at 690nm. Data are expressed as mean±standard deviations of at least eightreplicates per N/P ratio.

At the optimal N/P ratio (i.e., N/P=40), the DNA complex of cationicstar polymer CS-6 (Example 31) was not significantly toxic to HepG2 and4T1 cells (FIG. 7, bar chart). The viability of HepG2 and 4T1 cells wasmore than 85% after incubation with CS-6/DNA complex.

More than 80-90% cell viability was achieved after incubating HepG2cells with the DNA complexes of cationic graft polymers CG-5 (Example34), CG-6 (Example 35) and CG-7 (Example 36) at N/P=50, 20 and 20,respectively (FIG. 8, bar chart).

IX. Degradability of Dendritic Phenolic Esters

US patent application Ser. No. 13/077005 discloses G-2(OH)₁₂, shownbelow, which has 12 nucleophilic hydroxy groups capable of initiatingring opening polymerization of one or more cyclic carbonyl monomers(e.g., L-lactide, D-lactide, cyclic carbonates, lactones, and the like)to form a ROP star polymer comprising 12 polymer arms, such as SP12(Scheme A).

The starred bond represents the point of attachment of each R group (apolymer arm) to an oxygen in the SP12 structure. Thus, SP12 has 12polymer arms, each polymer arm independently comprising an innerhydrophobic poly(L-lactide) block attached to a core structure derivedfrom G-2(OH)₁₂, and a peripheral cationic polycarbonate block attachedto the poly(L-lactide) block. The order of the foregoing ring openingpolymerizations can be reversed, resulting in a star polymer having aperipheral hydrophobic block, as shown in SP13 below.

The L-lactide can be substituted with D-lactide in the ring openingpolymerization to form a star polymer having opposite stereospecificityif desired.

In the above structures of SP12 and SP13, n=10 and m=15.

The following example demonstrates the instability of the phenolic esterin D-1.

A vial was charged with D-1 (0.1 g, 0.153 mmol), DBU (0.0186 g, 0.122mmol), MeOH-d₄ (2 mL) and a stir bar. The reaction mixture was stirredat ambient temperature and observed every hour for degradation. After 1hour, significant loss of the phenolic ester group was observed.

The following example demonstrates the stability of the aliphatic esterin G-1(OH)₈ (Example 14 further above). A vial was charged withG-1(OH)₈, DBU (0.0186 g, 0.122 mmol), MeOH-d₄ (2.66 mL) and stir bar.The reaction mixture was stirred at ambient temp and observed every hourfor degradation. No change in the structure of G1-(OH)₈ was detectedafter 6 hours using NMR.

The MIC of SP12 was not determined because the phenolic ester groupshydrolyzed too easily. Instead, a linear ROP block copolymer wasprepared having the same polymer chain block structure as the R chain ofSP12. This linear block copolymer was tested for antimicrobial activityusing the same protocol as described above for unimolecular cationicstar polymers having a G′, G″, or G″′ core structure. The MIC against S.aureus was >500 mg/L for the linear block copolymer. This demonstratesthat core I′ groups containing repeat units that include carboxylicesters of phenolic alcohols are less preferred for I′ due to theirhydrolytic instability. This also demonstrates the inferiorantimicrobial properties of a linear ROP copolymer having a similarcationic arm structure as star polymers CS-1, CS-2, CS-3. That is, thequaternary amine group comprising a benzyl moiety was not sufficientalone to achieve enhanced antimicrobial properties. The greatestantimicrobial activity was observed with the cationic polymers having ahydrophobic branched core group and a quaternary amine group comprisinga benzyl moiety.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. When a range is used to express apossible value using two numerical limits X and Y (e.g., a concentrationof X ppm to Y ppm), unless otherwise stated the value can be X, Y, orany number between X and Y.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiments were chosen and described in order to best explain theprinciples of the invention and their practical application, and toenable others of ordinary skill in the art to understand the invention.

What is claimed is:
 1. A cationic graft polymer of formula (1):I′

P′]_(w′)  (1), wherein w′ is a positive number greater than or equal to3, I′ is a core comprising a multivalent linear aliphatic polycarbonatecomprising carbonate repeat units, the cationic graft polymer comprisesw′ independent peripheral monovalent cationic polymer chains P′, whereineach of the chains P′ is linked to a respective one of the carbonaterepeat units of I′, and each of the chains P′ is a homopolymer of acationic repeat unit selected from the group consisting of

and combinations thereof, wherein X^(⊖) is a negative chargedcounterion, and the cationic graft polymer is an effective antimicrobialagent against a Gram-positive microbe and/or a Gram-negative microbe. 2.The cationic graft polymer of claim 1, wherein I′ is a multivalentlinear aliphatic polyestercarbonate.
 3. The cationic graft polymer ofclaim 1, wherein I′ is free of carboxylic esters of phenolic alcohols.4. The cationic graft polymer of claim 1, wherein the cationic graftpolymer is biocompatible and/or enzymatically biodegradable.
 5. A methodof forming the cationic graft polymer of claim 1, comprising: forming amixture containing i) an organocatalyst, ii) an optional accelerator,iii) a solvent, iv) a cyclic carbonate monomer having the structure:

and v) an initiator comprising a linear aliphatic polycarbonatecomprising 3 or more side chain nucleophilic groups capable ofinitiating a ring opening polymerization (ROP); agitating the mixture,thereby forming an electrophilic polymer by ROP of the cyclic carbonylmonomer; optionally endcapping the electrophilic polymer, therebyforming an endcapped electrophilic polymer; and treating theelectrophilic polymer or the endcapped electrophilic polymer with atertiary amine selected from the group consisting of trimethylamine(TMA) and N,N,N′,N′-tetramethylethylenediamine (TMEDA), thereby formingthe cationic graft polymer.
 6. The method of claim 5, wherein theinitiator is a linear aliphatic polyestercarbonate comprising 3 or moreside chain nucleophilic groups capable of initiating a ring openingpolymerization (ROP).
 7. The method of claim 5, wherein the tertiaryamine is TMEDA.
 8. A method of killing a microbe comprising contactingthe microbe with the cationic polymer of claim
 1. 9. An injectablecomposition comprising an aqueous mixture of the cationic polymer ofclaim
 1. 10. A method of treating a cell comprising contacting the cellwith a composition comprising i) the cationic polymer of claim 1 and ii)a gene and/or a drug.
 11. An article, comprising the cationic graftpolymer of claim 1 disposed on a surface of a medical device.
 12. Thearticle of claim 11, wherein the medical device is selected from thegroup consisting of swabs, catheters, sutures, stents, bedpans, gloves,facial masks, absorbent pads, absorbent garments, internal absorbentdevices, insertable mechanical devices, wound dressings, and surgicalinstruments.
 13. A cationic graft polymer comprising i) a multivalentlinear aliphatic polycarbonate core I′ comprising 3 or more side chainsand ii) 3 or more independent monovalent cationic polymer chains P′,wherein each of the side chains is linked to a respective end unit ofone of the polymer chains P′, each of the polymer chains P′ is anindependent homopolymer of a cationic repeat unit selected from thegroup consisting of

wherein X^(⊖) is a negative charged counterion, and the cationic graftpolymer is an effective antimicrobial agent against a Gram-positivemicrobe and/or a Gram-negative microbe.
 14. The cationic polymer ofclaim 13, wherein the core I′ is a linear aliphatic polyestercarbonatecomprising 3 or more side chains.